Cyclooxygenase Inhibitor and Calcium Channel Antagonist Compositions and Methods for Use in Urological Procedures

ABSTRACT

Compositions of a cyclooxygenase inhibitor and a calcium channel antagonist in a liquid carrier. The composition may be administered the urinary tract during urological diagnostic, interventional, surgical and other medical procedures. One disclosed composition comprises ketoprofen and nifedipine in a liquid irrigation carrier, and includes a solubilizing agent, stabilizing agents and a buffering agent.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of U.S. patent applicationSer. No. 11/384,235, filed Mar. 17, 2006, now allowed, which claims thebenefit of U.S. Provisional Application No. 60/683,488, filed May 20,2005.

FIELD OF THE INVENTION

The present invention relates to pharmaceutical compositions foradministration to the urinary tract during urological diagnostic,interventional, surgical and other medical procedures and fortherapeutic treatment of urologic structures.

BACKGROUND OF THE INVENTION

Many urological procedures are now performed using minimally invasiveendoscopic (e.g., cystoscopic or uteroscopic) techniques. These includeexamination of the urethra, bladder and ureters, therapeutic treatmentsfor benign prostatic hypertrophy, removal or fragmentation of kidney andbladder stones, the placement of urethral or ureteral stents tofacilitate the passage of stones, the performance of biopsies and theexcision of tumors. While less invasive than open surgery, thesetechniques involve procedural irritation and trauma to the urinary tractleading to pain, inflammation and smooth muscle spasm. Postoperativelower urinary tract symptoms (LUTS) following urological proceduresoften include pain, hyperreflexia (unstable bladder contractions),urinary frequency, nocturia and urgency, and in some cases urinaryretention requiring prolonged catheterization.

For some surgical procedures, such as transurethral resection of theprostate (TURP), frequent urination and other symptoms resulting fromthe procedural irritation and inflammation may continue for a prolongedperiod, gradually resolving during the first six postoperative weeks.For urologic procedures employing a laser, postoperative complicationssuch as inflammation and muscle spasm may continue for several weeks.Patients are frequently prescribed oral anticholinergic medication toinhibit postoperative spasm and reduce the severity of unstablecontractions. However, not all patients respond adequately to thesedrugs, and side effects may lead to discontinuation of thesemedications.

Urological procedures are often performed with concurrent irrigation ofthe urinary tract, to remove blood and tissue debris so that a clearendoscopic field of view is maintained. Conventional irrigationsolutions include saline, lactated Ringer's, glycine, sorbitol, manitoland sorbitol/manitol. These conventional irrigation solutions do notcontain active pharmaceutical agents.

U.S. Pat. No. 5,858,017 to Demopulos, et al., the disclosure of which ishereby incorporated by reference, discloses surgical irrigationsolutions and methods for the inhibition of pain, inflammation and/orspasm. The use of irrigation solutions containing pain/inflammationinhibitors and anti-spasm agents during urological procedures in generaland during TURP specifically is disclosed, including five-drug andnine-drug combinations. This reference does not teach optimized pairingsof a pain/inflammation inhibitory agent with an anti-spasm agent forgiven urological procedures.

SUMMARY OF THE INVENTION

The present invention provides a locally deliverable composition forinhibiting pain/inflammation and spasm, comprising a combination ofketoprofen and a calcium channel antagonist in a carrier. Ketoprofen andthe calcium channel antagonist are each included in a therapeuticallyeffective amount such that the combination inhibits pain/inflammationand spasm at a site of local delivery.

In a further aspect of the present invention, a locally deliverablecomposition for inhibiting pain/inflammation and spasm comprises acombination of a cyclooxygenase inhibitor and a calcium channelantagonist, propyl gallate as a stabilizing agent and a liquid carrier.Each active agent is included in a therapeutically effective amount suchthat the combination inhibits pain/inflammation and spasm at a site oflocal delivery.

In a further aspect of the present invention, a locally deliverablecomposition for inhibiting pain/inflammation and spasm comprises acombination of a cyclooxygenase inhibitor and a calcium channelantagonist an aqueous liquid carrier, a cosolvent, at least onestabilizing agent and a buffer. Each active agent is included in atherapeutically effective amount such that the combination inhibitspain/inflammation and spasm at a site of local delivery.

A further aspect of the present invention provides a method ofinhibiting pain/inflammation and spasm in the urinary tract, comprisingdelivering to the urinary tract a composition including a combination ofketoprofen and a calcium channel antagonist in a carrier. Ketoprofen andthe calcium channel antagonist are each included in a therapeuticallyeffective amount such that the combination inhibits pain/inflammationand spasm in the urinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain/inflammation and spasm in the urinary tract during adiagnostic, interventional, surgical or other medical urologicalprocedure, comprising periprocedurally delivering to the urinary tractduring a urological procedure a composition including a combination ofketoprofen and nifedipine in a carrier. Ketoprofen and nifedipine areeach included in a therapeutically effective amount such that thecombination inhibits pain/inflammation and spasm in the urinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain/inflammation and spasm in the urinary tract during aurological procedure, comprising periprocedurally delivering to theurinary tract during a ureteroscopic procedure a composition including acombination of a cyclooxygenase inhibitor and a calcium channelantagonist in a carrier. The cyclooxygenase inhibitor and the calciumchannel antagonist are each included in a therapeutically effectiveamount such that the combination inhibits pain/inflammation and spasm inthe urinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain/inflammation and spasm in the urinary tract during aurological procedure, comprising periprocedurally delivering to theurinary tract during a procedure to remove, fragment or dislodge akidney or bladder stone a composition including a combination of acyclooxygenase inhibitor and a calcium channel antagonist in a carrier.The cyclooxygenase inhibitor and the calcium channel antagonist are eachincluded in a therapeutically effective amount such that the combinationinhibits pain/inflammation and spasm in the urinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain/inflammation and spasm in the urinary tract during aurological procedure, comprising periprocedurally delivering to aurologic structure during a procedure that causes thermal injury tourinary tract tissue a composition including a combination of acyclooxygenase inhibitor and a calcium channel antagonist in a carrier.The cyclooxygenase inhibitor and the calcium channel antagonist are eachincluded in a therapeutically effective amount such that the combinationinhibits pain/inflammation and spasm in the urinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain, inflammation and/or spasm in the urinary tract during aurological procedure, comprising periprocedurally delivering to aurologic structure during a ureteroscopic procedure a compositionincluding a combination of a plurality of agents that inhibitpain/inflammation and/or spasm in a carrier. Each agent is included in atherapeutically effective amount such that the combination inhibitspain/inflammation and/or spasm in the urinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain, inflammation and/or spasm in the urinary tract during aurological procedure, comprising periprocedurally delivering to aurologic structure during a procedure to remove, fragment or dislodge akidney or bladder stone a composition including a combination of aplurality of agents that inhibit pain/inflammation and/or spasm in acarrier. Each agent is included in a therapeutically effective amountsuch that the combination inhibits pain/inflammation and/or spasm in theurinary tract.

A still further aspect of the present invention provides a method ofinhibiting pain, inflammation and/or spasm in the urinary tract during aurological procedure, comprising periprocedurally delivering to aurologic structure during a procedure that causes thermal injury tourinary tract tissue a composition including a combination of aplurality of agents that inhibit pain/inflammation and/or spasm in acarrier. Each agent is included in a therapeutically effective amountsuch that the combination inhibits pain/inflammation and/or spasm in theurinary tract.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will now be described in greater detail, by way ofexample, with reference to the accompanying drawings in which:

FIG. 1 provides a model for action of prostaglandin activity.

FIG. 2 illustrates the bradykinin and substance P cumulativeconcentration-response curves obtained from normal animals in Example I.

FIG. 3A illustrates bradykinin concentration-response curves produced inthe presence of 0.25, 1.0, 2.5 and 10 μM ketoprofen from Example I; FIG.3B illustrates the Schild plot for pA2 analysis of ketoprofen fromExample I.

FIG. 4A demonstrates that bradykinin rapidly induces the formation ofPGE₂ in rat bladder tissue strips tested in Example I within the firstminutes of stimulation and reaches a maximum within 30 minutes, with at_(1/2) for formation of about 7.5 minutes.

FIG. 4B illustrates the rapid kinetics of PGE₂ formation detected withinminutes in Example I.

FIG. 5A illustrates that intravenous aspirin (10 mg/kg) produced agradual time-dependent inhibition of the acetic acid induced reductionin the intercontraction interval (ICI), and FIG. 5B illustrates theparallel changes in bladder capacity, from Example I.

FIG. 6 shows the effect of increasing concentrations of nifedipine oncontractility of rat bladder strips from Example II.

FIG. 7 shows the combined effect of nifedipine (0.1 μM) and ketoprofen(0.3-3.0 μM) on bradykinin-stimulated contractility of rat bladderstrips from Example III.

FIG. 8 shows the combined effect of nifedipine (0.3 μM) and ketoprofen(0.3-3.0 μM) on bradykinin-stimulated contractility of rat bladderstrips from Example III.

FIG. 9 shows the combined effect of nifedipine (1.0 μM) and ketoprofen(0.3-3.0 μM) on bradykinin-stimulated contractility of rat bladderstrips from Example III.

FIG. 10 illustrates the concentration-response surface (reduced model)of individual tension values from dose response curves corresponding to30 μM bradykinin-induced tension in rat bladder strips from Example III.

FIG. 11 shows the effect of ketoprofen (10 μM) and nifedipine (1 μM),individually, on multiple agonist-stimulated tension in rat bladdertissue strips from Example IV.

FIG. 12 shows the effect of ketoprofen (10 μM) and nifedipine (1 μM),individually, on bradykinin-stimulated PGE₂ release from rat bladdertissue strips from Example IV.

FIG. 13 shows a rat bladder cystometry tracing demonstrating the effectof acetic acid perfused as described in Example V.

FIG. 14 demonstrates the effect of ketoprofen pretreatment on aceticacid-induced bladder hyperactivity from Example V.

FIG. 15 demonstrates the effect of nifedipine pretreatment on aceticacid-induced bladder hyperactivity from Example V.

FIG. 16 illustrates mean ketoprofen plasma levels for rats treated withketoprofen or a combination of ketoprofen and nifedipine in thepharmacokinetic study of Example VI.

FIG. 17 illustrates mean nifedipine plasma levels for rats treated withnifedipine or a combination of ketoprofen and nifedipine in thepharmacokinetic study of Example VI.

FIG. 18 illustrates the effects of nifedipine, ketoprofen and acombination of nifedipine and ketoprofen on PGE₂ in rat bladders fromthe pharmacokinetic study of Example VI.

FIG. 19 shows a chromatogram of a nifedipine and ketoprofen formulationF1 in accordance with Example VIII after having been stressed at 60° C.for 1 month.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides methods and compositions for inhibitingpain, inflammation and/or spasm during urological procedures by locallydelivering such compositions to structures of the urological tractduring the procedure. The compositions include at least one agent thatis a pain/inflammation inhibitory agent or a spasm inhibitory agent, orthat acts to inhibit both pain/inflammation and spasm. Preferably, thecompositions and methods of the present invention include two or morepain/inflammation inhibitory or spasm inhibitory agents that act ondifferent molecular targets (i.e., enzymes, receptors or ion channels)or that act through different mechanisms of action. More preferably, thecompositions of the present invention include at least onepain/inflammation inhibitory agent and at least one spasm inhibitoryagent.

As used herein, the term “pain/inflammation inhibitory agent” includesanalgesic agents (i.e., antinociceptive agents), non-steroidal agentsthat inhibit inflammation [including both “non-steroidalanti-inflammatory drugs” (i.e., NSAIDS or cyclooxygenase inhibitors) andother agents that are not steroidal that act to inhibit inflammation],corticosteroids and local anesthetics.

As used herein, the term “spasm inhibitory agent” includes agents thatinhibit spasm or contraction of smooth muscle tissue and agents thatinhibit spasm or contraction of other muscle tissue associated with theurinary tract (e.g., prostatic muscle tissue).

Another aspect of the present invention is directed to theperiprocedural delivery to the urinary tract of a cyclooxygenase (COX)inhibitor, suitably a non-selective COX-1/COX-2 inhibitor, preferably anon-selective COX-1/COX-2 inhibitor that is a propionic acid derivative,more preferably ketoprofen, alone or with at least one additional agentthat inhibits pain/inflammation and/or that inhibits spasm, such as acalcium channel antagonist.

Another aspect of the present invention is directed to theperiprocedural delivery to the urinary tract of a calcium channelantagonist (i.e., a calcium channel blocker), suitably an L-type calciumantagonist, preferably a dihydropyridine calcium channel antagonist,more preferably nifedipine, alone or with at least one additional agentthat inhibits pain/inflammation and/or that inhibits spasm, such as aCOX inhibitor.

Another aspect of the present invention is directed to theperiprocedural delivery to the urinary tract of a combination of a COXinhibitor and a calcium channel antagonist, preferably a non-selectiveCOX-1/COX-2 inhibitor in combination with an L-type calcium antagonist,more preferably ketoprofen in combination with nifedipine. Ketoprofenand nifedipine have been found by the present inventors to providegreater than additive or synergistic results in the inhibition ofbladder spasm, as described in the examples below.

One aspect of the present invention entails the local delivery of thecompositions of the present invention to the bladder, ureter, urethra,or other urinary tract structures to inhibit pain, inflammation and/orsmooth muscle spasm during urological therapeutic, diagnostic,interventional, surgical and other medical procedures.

As used herein, the terms “urinary tract” and “urinary system” refer tothe kidneys, ureters, bladder, urethra and associated nerves, bloodvessels and muscles. The term “lower urinary tract” refers to thebladder and urethra and associated nerves, blood vessels and muscles.

A further aspect of the present invention entails the local delivery ofthe compositions of the present invention to urinary tract structures toreduce postoperative irritative voiding symptoms (e.g., void frequency,nocturia, urgency), pain and/or other lower urinary tract symptomsfollowing such urological procedures.

A further aspect of the present invention entails the local delivery ofthe compositions of the present invention to urinary tract structures toimprove postoperative urinary function (e.g., decrease undesirableurinary retention) following such urological procedures.

The compositions of the present invention are suitably delivered to theurinary tract before, during and/or after urological procedures, i.e.,before (pre-) procedurally, during (intra-) procedurally, after (post-)procedurally, pre- and intraprocedurally, pre- and postprocedurally,intra- and postprocedurally or pre-, intra- and postprocedurally.

Preferably, the compositions of the present invention are locallydelivered to the urinary tract “periprocedurally”, which as used hereinmeans intraprocedurally, pre- and intraprocedurally, intra- andpostprocedurally or pre-, intra- and postprocedurally. Periproceduraldelivery may be either continuous or intermittent during the procedure.Preferably, the compositions of the present invention are delivered“continuously” during the procedure, which as used herein means deliveryso as to maintain an approximately constant concentration of activeagent(s) at the local delivery site. When delivered periprocedurallyduring a surgical procedure, the term “perioperatively” may be usedinterchangeably with periprocedurally herein. Preferably, thecompositions of the present invention are delivered periprocedurelyduring the period of time when surgical or other procedural trauma andirritation is being incurred by urinary tract tissue.

“Local” delivery of the compositions of the present invention to theurinary tract as used herein refers to delivery of the compositionsdirectly to one or more structures of the urinary tract. The therapeuticagent(s) contained in the locally delivered compositions are not subjectto first and/or second pass metabolism before reaching the local site ofintended therapeutic (e.g., inhibitory) effect, in contrast tosystemically delivered drugs.

Pathophysiologic Effects of Urological Procedures

The trauma of urological procedures results in an acute, localizedinflammatory response in the associated urological structures.Inflammation is associated with a complex pattern of biochemical andcellular processes occurring at the local site, involvingpositive-feedback interactions between the peripheral nervous system,immune cells, the local vasculature and the central nervous system. Theinflammatory response to procedural trauma in the urinary tract includescytokine release, inflammatory cell migration, edema, pain andhyperalgesia.

In response to tissue injury, numerous local mediators are rapidlyreleased, which result in nociceptive stimulation of sensory C-fibers.The inflammatory response triggered by peripheral injury shows that, inaddition to cytokines, small G-protein receptor-linked inflammatorymediators also modulate the rapid pathophysiological response of thebladder and urethra. In models of urinary bladder inflammation,bradykinin, histamine, substance P(SP), leukotrienes and prostaglandinshave been found to be released from the bladder. Lecci, A., et al.,Pharmacological Analysis of the Local and Reflex Responses to Bradykininon Rat Urinary Bladder Motility in Vivo, Br. J. Pharmacol., 114:708-14(1995); Lecci, A., et al., Capsaicin Pretreatment Does Not Alter RatUrinary Bladder Motor Responses Induced by a Kinin B1 Receptor AgonistAfter Endotoxin Treatment, Neurosci. Lett. 262:73-76 (1999); Vasko, M.,et al., Prostaglandin E2 Enhances Bradykinin-Stimulated Release ofNeuropeptides from Rat Sensory Neurons in Culture, J Neurosci.14:4987-97 (1994). Certain inflammatory mediators, such asprostaglandins and kinins, activate and sensitize C-fibers throughinteraction with specific receptors on nerve terminals. Otherinflammatory mediators that have been described in the lower urinarytract include tachykinins and ATP (from C-fibers) (Maggi, C., et al.,Tachkykinin Antagonists and Capsaicin-Induced Contraction of the RatIsolated Urinary Bladder: Evidence for Tachykinin-MediatedCotransmission, Br. J. Pharmacol. 103:1535-41 (1991), CGRP (fromC-fibers), serotonin (from mast cells and platelets), and endothelin.Maggi, C., et al., Contractile Responses of the Human Urinary Bladder,Renal Pelvis and Renal Artery to Endothelins and Sarafotoxin S6b, Gen.Pharmacol. 21:247-49 (1990). These mediators operate together in asynergistic manner to increase postsurgical hyperalgesia, inflammationand muscle spasm. The number of mediators involved in the responseunderscores the multifactorial origin of the pain and inflammationprocess.

The immediate activation of the sensory nerves (primary hyperalgesia)triggers a cascade of processes that involves alterations in the localvasculature, and influences muscle contractility. Capsaicin-sensitiveafferent fiber stimulation elicits a local efferent response, which ischaracterized by release of neuropeptides (tachykinins and CGRP) fromnerve endings. This release produces a number of local responses, whichare part of the pathophysiological effects in the lower urinary tract.These include: (1) direct effects of released neurotransmitters onsmooth muscle contraction; (2) changes in microvascular permeabilityresulting in plasma extravasation and edema of the bladder, urethra andprostate; (3) infiltration of immune cells; and (4) sensitization ofnociceptors (secondary hyperalgesia) resulting in increased pain. Theconsequences of these processes can affect normal bladder capacity andfrequency of micturition, and often result in hypersensitivity, pain andsmooth muscle spasm.

The pathophysiologic response to procedural trauma of the urinary tractinvolves a complex cascade of molecular signaling and biochemicalchanges resulting in inflammation, pain, spasm and lower urinary tractsymptoms. These are preferably addressed in accordance with the methodsand compositions of the present invention by locally andperiprocedurally delivering a combination of pharmacologic agents actingon multiple molecular targets to inhibit pain, inflammation and/orspasm. Preferred agents include cyclooxygenase inhibitors and calciumchannel antagonists, more preferably in combination.

Cyclooxygenase Inhibitors

Prostaglandins are produced throughout the lower urinary tract and playa role in neurotransmission, bladder contractility and inflammatoryresponses. Human bladder mucosa has been found to contain several typesof prostaglandins, which have been shown to contract the human detrusor.Prostaglandin E₂ (PGE₂) is a potent mediator of pain and edema, and theexogenous administration of PGE₂ induces contractile responses ininflamed bladders. Intravesical PGE₂ produces both urgency andinvoluntary bladder contractions. Lepor, H., The Pathophysiology ofLower Urinary Tract Symptoms in the Ageing Male Population, Br. J Urol.,81 Suppl 1:29-33 (1998); Maggi, C., et al., Prostanoids Modulate ReflexMicturition by Acting Through Capsaicin-Sensitive Afferents, Eur. J.Pharmacol. 145: 105-12 (1988). PGE₂ given intravesically may stimulatemicturition by releasing tachykinins from nerves in and/or immediatelybelow the urothelium. Ishizuka, O., et al., Prostaglandin E2-InducedBladder Hyperactivity in Normal, Conscious Rats: Involvement ofTachykinins?, J Urol. 153:2034-38 (1995). Prostanoids may, via releaseof tachykinins, contribute to both urge and bladder hyperactivity seenin inflammatory conditions of the lower urinary tract. While not wishingto be limited by theory, these actions are most likely mediated throughactivation of specific prostanoid receptor subtypes (EP1R) located onC-fibers and on bladder smooth muscle (FIG. 1).

In the inflamed bladder, the basal production of PGE₂ is significantlyhigher than in control conditions. A number of inflammatory mediatorsacting through GPCR pathways that are linked to the production ofarachidonic acid may up-regulate prostaglandin levels in the mucosa andvascular endothelium. Bradykinin is a well-established mediator ofinflammation, and bradykinin receptor agonists stimulate greater PGE₂production in inflamed bladders than in control bladders. Topicalapplication of bradykinin activates bladder sensory nerves. Lecci, A.,et al., Kinin B1 Receptor-Mediated Motor Responses in Normal or InflamedRat Urinary Bladder in Vivo, Regul. Pept. 80:41-47 (1999); Maggi, C., etal., Multiple Mechanisms in the Motor Responses of the Guinea-PigIsolated Urinary Bladder to Bradykinin, Br. J. Pharmacol. 98:619-29(1989). Contractile responses elicited by the selective B1 and B2receptor agonists tested in isolated rat urinary bladder strips showedthat the contractile responses to a selective B1 agonist were alsopotentiated in inflamed bladders. The role of bradykinin in reflexvoiding has also been investigated in normal rats using continuousinfusion cystometry. Infusion of bradykinin produced a significantdecrease in the intercontraction interval (ICI) between voiding eventsand an increase in bladder contraction amplitude that is completelyblocked by a B2 receptor antagonist.

Microvascular leakage induced by administration of substance P actingthrough the NK1 receptor also involves the release of cyclooxygenasemetabolites of arachidonic acid. Abelli, L., et al., MicrovascularLeakage Induced by Substance P in Rat Urinary Bladder: Involvement ofCyclo-oxygenase Metabolites of Arachidonic Acid, J. Auton. Pharmacol.12:269-76 (1992). These findings demonstrate that distinct inflammatorymediators act through independent receptor mechanisms to trigger theproduction of prostaglandins. NSAIDs that act at a common targetdownstream of multiple GPCRs to inhibit COX-1/COX-2 have the capacity toblock the formation of prostaglandins derived from multipleproinflammatory mediators.

A number of studies have shown that both COX-1 and COX-2 are involved inthe production of PGE₂ during tissue trauma and the acute inflammatoryresponse. Martinez, R., et al., Involvement of PeripheralCyclooxygenase-1 and Cyclooxygenase-2 in Inflammatory Pain, J PharmPharmacol. 54:405-412 (2002); Mazario, J, et al., Cyclooxygenase-1 vs.Cyclooxygenase-2 Inhibitors in the Induction of Antinociception inRodent Withdrawal Reflexes, Neuropharmacology. 40:937-946 (2001);Tones-Lopez, J., et al., Comparison of the Antinociceptive Effect ofCelecoxib, Diclofenac and Resveratrol in the Formalin Test, Life Sci.70:1669-1676 (2002). In normal bladders, activation of B2 receptorsevokes bladder contraction mediated by COX-1 activity, whereas COX-2activity is involved in production of PGE₂ driven through stimulation ofB1 receptors only. COX-2 is the major isoform that is rapidly expressedand dramatically up-regulated during bladder inflammation. It isbelieved to be responsible for the high levels of prostanoids releasedduring acute and chronic inflammation of the bladder. COX-2 isup-regulated in response to proinflammatory cytokines and bladdertreatment with either endotoxin or cyclophosphamide. Both COX isozymesare therefore suitable molecular targets for the drug compositions ofthe present invention.

An aspect of the present invention is directed to therapeuticcompositions including a cyclooxygenase inhibitor in a carrier suitablefor local delivery to urologic structures in the urinary tract. Toachieve maximal inhibition of prostaglandin synthesis at sites of acuteinflammation, it is believed desirable to inhibit both COX isoenzymes.

The COX inhibitor is therefore preferably non-selective with respect toactivity at COX-1 and COX-2, which for purposes of the present inventionmay be defined as an agent for which the ratio of (a) the concentrationof the agent effective for the inhibition of 50% (IC50) of the activityof COX-1 relative to (b) the IC50 for the inhibition of the activity ofCOX-2 is greater than or equal to 0.1 and less than or equal to 10.0,and more preferably is greater than or equal to 0.1 and less than orequal to 1.0. Suitable assays for determining COX-1 and COX-2 inhibitoryeffect are disclosed in Riendau, D., et al., Comparison of theCyclooxygenase-1 Inhibitory Properties of Nonsteroidal Anti-inflammatoryDrugs (NSAIDs) and Selective COX-2 Inhibitors, Using SensitiveMicrosomal and Platelet Assays, Can. J. Physiol. Pharmacol. 75:1088-1095(1997).

Suitable non-selective COX-1/COX-2 inhibitors include, for purposes ofillustration, salicylic acid derivatives including aspirin, sodiumsalicylate, choline magnesium trisalicylate, salsalate, diflunisal,sulfasalazine and olsalazine, para-aminophenol derivatives such asacetaminophen, indole and indene acetic acids such as indomethacin andsulindac, heteroaryl acetic acids including tolmetin, diclofenac andketerolac, arylpropionic acids including ibuprofen, naproxen,flurbiprofen, ketoprofen, fenoprofen and oxaprozin, anthranilic acids(fenamates) including mefanamic acid and meclofenamic acid, enolic acidsincluding oxicams such as piroxicam and meloxicam and alkanones such asnabumetone, as well as pharmaceutically effective esters, salts,isomers, conjugates and prodrugs thereof.

Still more preferably, the non-selective COX-1/COX-2 inhibitor is anarylpropionic acid, i.e., a propionic acid derivative, such asketoprofen, dexketoprofen, ibuprofen, naproxen, flurbiprofen, fenoprofenand oxaprozin. Most preferably, the agent is ketoprofen.

In another aspect of the invention, the non-selective COX-1/COX-2inhibitor used in the compositions and methods of the present inventionis selected as having an IC50 for the inhibition of bradykinin-inducedbladder smooth-muscle strip contractility (as determined by the bladdercontractility model described herein below) of less than or equal to 100μM, preferably less than or equal to 25 μM, more preferably less than orequal to 5 μM, still more preferably less than 2 μM.

In a further aspect of the invention, the non-selective COX-1/COX-2inhibitor used in the compositions and methods of the present inventionis selected as having an IC50 for the inhibition of bradykinin-inducedprostaglandin E₂ (PGE₂) (as determined by the PGE₂ bladder tissueanalysis model described herein below) of less than or equal to 100 μM,preferably less than or equal to 25 μM, more preferably less than orequal to 5 μM, still more preferably less than 2 μM.

In a still further aspect of the invention, the non-selectiveCOX-1/COX-2 inhibitor used in the compositions and methods of thepresent invention is selected as having (a) an IC50 for the inhibitionof bradykinin-induced bladder smooth-muscle strip contractility (asdetermined by the bladder contractility model described herein below) ofless than or equal to 100 μM, preferably less than or equal to 25 μM,more preferably less than or equal to 5 μM, still more preferably lessthan 2 μM, and (b) an IC50 for the inhibition of bradykinin-induced PGE₂(as determined by the PGE₂ bladder tissue analysis model describedherein below) of less than or equal to 100 μM, preferably less than orequal to 25 μM, more preferably less that or equal to 5 μM, still morepreferably less than 2 μM.

The above noted IC50 concentrations are not to be interpreted aslimitations on drug concentrations in the compositions of the presentinvention, which may suitably be determined by the concentrations neededto approach maximal effectiveness and thus may be higher than the IC50levels.

In a still further aspect of the invention, the non-selectiveCOX-1/COX-2 inhibitor used in the compositions and methods of thepresent invention is selected as having a pA₂ (antagonist potency) ofgreater than or equal to 7, wherein pA₂ is the negative logarithm of theconcentration of antagonist that would produce a 2-fold shift in theconcentration response curve for an agonist, and is a logarithmicmeasure of the potency of an antagonist. This potency corresponds to anequilibrium dissociation constant K_(D) of less than or equal to 100 nM.

In a still further aspect of the invention, the non-selectiveCOX-1/COX-2 inhibitor used in the compositions and methods of thepresent invention exhibits 50% of maximal inhibitory response in lessthan or equal to 10 minutes in a kinetic study of bradykinin-stimulatedPGE₂ response in the PGE₂ bladder tissue analysis model described hereinbelow.

Ketoprofen

Unless used in a context also referring to its isomer, references hereinto the use of ketoprofen (i.e., m-benzoylhydratropic acid or3-benzoyl-α-methylbenzeneacetic acid) in the present invention are to beunderstood to also include pharmaceutically acceptable isomers thereof,including its racemic S-(+)-enantiomer, dexketoprofen, pharmaceuticallyacceptable salts or esters thereof, and pharmaceutically acceptableprodrugs or conjugates thereof. Ketoprofen is a preferred COX inhibitorfor use in the present invention.

Ketoprofen exhibits potent anti-inflammatory, analgesic, and antipyreticactions that are associated with the inhibition of prostaglandinsynthesis and antagonism of the effects of bradykinin. Ketoprofennon-selectively inhibits the activity of COX-1 and COX-2, which resultsin the blockade of prostaglandin production, particularly that of PGE₂,preventing the development of hyperalgesia. Ketoprofen has an IC₅₀ valueof 4-8 nM in a non-selective COX assay, being functionally 6-12 timesmore potent than other NSAIDs evaluated (e.g., naproxen orindomethacin). Kantor, T., Ketoprofen: A review of its Pharmacologic andClinical Properties, Pharmacotherapy 6:93-103 (1986). Ketoprofen alsohas functional bradykinin antagonist activity, its effects being eighttimes greater than those seen with the classical NSAID, indomethacin.Julou, L., et al., Ketoprofen (19.583 R.P.)(2-(3-Benzoylphenyl)-propionic acid). Main PharmacologicalProperties—Outline of Toxicological and Pharmacokinetic Data, Scand JRheumatol Suppl. 0:33-44 (1976).

In addition to inhibiting cyclooxygenase, ketoprofen is believed tooffer the additional anti-inflammatory benefit of inhibitinglipoxygenase. Ketoprofen has also been found to synergise withnifedipine in the inhibition of bladder spasm, as discussed in greaterdetail in the examples below.

Calcium Channel Antagonists

Multiple inflammatory mediators, including bradykinin, are released intothe bladder in response to tissue injury, which can trigger smoothmuscle contraction and spasm. The tone of the urinary bladder smoothmuscle is regulated by numerous contraction-promoting receptor systems.They include well established systems such as muscarinic, purinergic andtachykinin receptors [Anderson, K., et al., Pharmacology of the LowerUrinary Tract: Basis for Current and Future Treatments for UrinaryIncontinence Pharmacol Rev. 56:581-631 (2004)], and also includeendothelin receptors [Afiatpour, P., et al., Development Changes in theFunctional, Biochemical and Molecular Properties of Rat BladderEndothelin Receptors, Naunyn Schmiedebergs Arch. Pharmacol. 367:462-72(2003)], protease-activated receptors and bradykinin receptors [Kubota,Y., et al., Role of Mitochondria in the Generation of SpontaneousActivity in Detrusor Smooth Muscles of the Guinea Pig Bladder, J. Urol.170:628-33 (2003); Trevisani, M., et al., Evidence for In VitroExpression of B1 Receptor in the Mouse Trachea and Urinary Bladder, Br.J. Pharmacol. 126:1293-1300 (1999)]. Because many of these receptors areprototypically coupled via G_(q) proteins to the activation of aphospholipase C (PLC), it is likely that bladder contraction elicited bysuch receptors is partly mediated by PLC-linked mobilization of Ca²⁺from intracellular stores [Ouslander, J. G., Management of OveractiveBladder, N. Engl. J. Med., 350:786-99 (2004)].

Neurally mediated contractions of the bladder and urethral smooth musclerequire mobilization of intracellular Ca²⁺ as well as an influx ofextracellular Ca²⁺. Ca²⁺ entry through L-type calcium channels cancontribute to muscle contractions by triggering the intracellularrelease of Ca²⁺, which opens ryanodine-sensitive Ca²⁺ release channelsin the sarcoplasmic reticulum. Opening of L-type calcium channels inbladder muscle also serves to replace intracellular Ca²⁺ stores aftercontraction. Recent studies conclude that muscarinic receptor subtypesignaling mediated via carbachol-induced contraction of rat bladderslargely depends on Ca²⁺ entry through L-type calcium channels and,perhaps, PLD, PLA₂ and store-operated Ca²⁺ channels. Schneider, T., etal., Signal Transduction Underlying Carbachol-Induced Contraction of RatUrinary Bladder: I. Phospholipases and Ca2+sources, J Pharmacol Exp Ther(2003). Thus, blockade of L-type Ca²⁺ channels has the potential todepress neural, urothelial and smooth muscle evoked contractions ofbladder strips mediated by a multiplicity of endogenous GPCR agonists.The L-type calcium channel represents a point of integration for theconvergence of multiple inflammatory mediators that can lead tohyperactive smooth muscle contractility.

Ca²⁺ channels located in afferent and efferent nerve terminals in thelower urinary tract are also important for regulation ofneurotransmitter release. de Groat, W., et al., Pharmacology of theLower Urinary Tract, Annu Rev. Pharmacol Toxicol. 41:691-721 (2001). Anumber of active agents produce Ca²⁺ influx and transmitter release fromthe peripheral nerve endings of capsaicin-sensitive afferent neuronsthrough voltage-sensitive Ca²⁺ channels. Under certain conditions,L-type Ca²⁺ channels can also contribute to transmitter release.

The significant role of the L-type Ca²⁺ channel in the initiation ofsmooth muscle contraction makes this channel a potential therapeutictarget for the treatment of lower urinary tract problems that involvehyperactivity or spasm of smooth muscle tissues. In the presence ofinflammatory mediators, signaling through these same channels maymediate bladder hyperactivity and spasm.

An aspect of the present invention is thus directed to therapeuticcompositions including a calcium channel antagonist in a carriersuitable for delivery to urologic structures in the urinary tract. Thecalcium channel antagonist is preferably an L-type calcium channelantagonist, such as verapamil, diltiazem, bepridil, mibefradil,nifedipine, nicardipine, isradipine, amlodipine, felodipine, nisoldipineand nimodipine, as well as pharmaceutically effective esters, salts,isomers, conjugates and prodrugs thereof. Still more preferably, thecalcium channel antagonist is a dihydropyridine, such as nifedipine,nicardipine, isradipine, amlodipine, felodipine, nisoldipine andnimodipine, as well as pharmaceutically effective esters, salts,isomers, conjugates and prodrugs thereof. Most suitably, the agent isnifedipine.

Nifedipine

References herein to nifedipine,1,4-dihydro-2,6-dimethyl-4-(2-nitrophenyl)-3,5-pyridinedicarboxylic aciddimethyl ester, are to be understood to also include pharmaceuticallyacceptable isomers thereof, pharmaceutically acceptable salts or estersthereof, and pharmaceutically acceptable prodrugs or conjugates thereof.Nifedipine is a preferred calcium channel antagonist for use in thepresent invention.

Nifedipine is a member of the dihydropyridine class of calcium channelantagonists with pharmacological specificity for the L-type channel(alternatively termed the Cav1.2 α-subunit). Nifedipine has a rapidonset of action (less than 10 minutes), which is desirable for use inurological procedures, and as such is more preferred than certainclosely related dihydropryidine calcium channel antagonists (e.g.,amlodipine) that require longer periods for initial action. The time toresponse for steady-state inhibition of muscle contraction ideally occurwithin 10-15 minutes of initial local drug delivery, and nifedipinefulfills this criterion.

Carriers

The pain/inflammation and/or spasm agents of the present invention aresuitably delivered in solution or in suspension in a liquid carrier,which as used herein is intended to encompass biocompatible solvents,suspensions, polymerizable and non-polymerizable gels, pastes andsalves. Preferably, the carrier is an aqueous irrigation solution thatmay or may not include physiologic electrolytes, such as saline,distilled water, lactated Ringer's solution, glycine solutions, sorbitolsolutions, manitol solutions or sorbital/manitol solutions. The carriermay also include a sustained release delivery vehicle, such asmicroparticles, microspheres or nanoparticles composed of proteins,liposomes, carbohydrates, synthetic organic compounds, or inorganiccompounds.

The compositions of the present invention may also be coated on ureteraland urethral stents, catheters, radioactive seeds, seed spacers andother implantable devices and on surgical instruments, for localdelivery from such devices and instruments into the urinary tract asfurther described below. Polymers that may be suitably employed to forma drug impregnated stent or other implantable device include, by way ofnon-limiting example, poly(D,L-lactic acid) (PDLLA),poly(lactide-co-glyocide) (PLGA), poly(L-lactic acid) (PLLA),poly(glycolic acid), poly(6-hydroxycaproic acid), poly(5-hydroxyvalericacid), poly(4-hydroxybutyric acid), poly(ethylene glycol), poly(ethyleneoxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO,Pluronics™) block copolymers, and copolymers and blends of the above.

Suitable materials for use in producing drug coated stents, catheters,other implantable devices and instruments include biodegradable polymersand polymeric hydrogels, such as by way of nonlimiting example,Pluronics™ triblock copolymers, PLLAs or their copolyesters,poly(glycolic acid) or their copolyesters, poly(ethyleneoxide)-cyclodextrin(polyrotaxan)hydrogels,poly[(R)-3-hydroxybutyrate]-poly(ethylene oxide)-cyclodextrin hydrogels,cellulose acetate, cellulose acetate butyrate, cellulose acetatepropionate, and cellulose nitrate; polyurethane resins, including thereaction product of 2,4-tolylene diisocyanate, 4,4′-diphenylmethanediisocyanate, polymethylenepolyphenyl isocyanate, or 1,5-napthylenediisocyanate with 1,2-polypropylene glycol, polytetramethylene etherglycol, 1,4-butanediol, 1,4-butylene glycol, 1,3-butylene glycol,poly(1,4-oxybutylene)glycol, caprolactone, adipic acid esters, phthalicanhydride, ethylene glycol, 1,3-butylene glycol, 1,4-butylene glycol ordiethylene glycol; acrylic polymers such as ethyl and methyl acrylateand methacrylate; condensation polymers such as those produced bysulfonoamides such as toluenesulfonamide and aldehydes such asformaldehyde; isocyanate compounds; poly(ortho esters);poly(anhydrides); polyamides; polycyanoacrylates, poly(amino acids),polycarbonate), cross-linked poly(vinyl alcohol), polyacetals,polycaprolactone. In addition to these biodegradable polymers, suitablenon-biodegradable polymers include polyacrylates, polystyrenes,polyvinyl chloride, ethylene-vinyl acetate copolymers, polyvinylfluoride, poly(vinyl imidazole) and chlorosulphonated polyolefins.

The pain/inflammation and/or spasm inhibitory compositions of thepresent invention can also include excipients or adjuvants for enhanceduptake, release, solubility and stability. Aspects of formulating thecompositions of the present invention are discussed below.

Additional Agents

The cyclooxygenase inhibitor, calcium channel antagonist or combinationcyclooxygenase inhibitor plus calcium channel antagonist compositions ofthe present invention may include alternate or additional agents thatinhibit pain, inflammation and/or spasm. Suitable agents include thosedisclosed in U.S. Pat. No. 5,858,017 to Demopulos.

In particular, suitable alternate or additionalanti-inflammation/anti-pain agents include serotonin receptorantagonists, (e.g., amitriptyline, imipramine, trazodone, desipramine,ketanserin, tropisetron, metoclopramide, cisapride, ondansetron,yohimbine, GR127935, methiothepin), serotonin receptor agonists (e.g.,buspirone, sumatriptan, dihydroergotamine, ergonovine), histaminereceptor antagonists (e.g., promethazine, diphenhydramine,amitriptyline, terfenadine, mepyramine (pyrilamine), tripolidine),bradykinin receptor antagonists (e.g., [Leu⁸] des-Arg⁹-BK, [des-Arg¹⁰]derivative of HOE 140, [leu⁹] [des-Arg¹⁰] kalliden, [D-Phe⁷]-BK, NPC349, NPC 567, HOE 140), kallikrien inhibitors (e.g., aprotinin),tachykinin receptor antagonists, including neurokinin₁ receptor subtypeantagonists (e.g., GR 82334, CP 96.345, RP 67580) and neurokininreceptor subtype antagonists (e.g., MEN 10.627, L 659.877, (±)-SR48968), calcitonin gene-related peptide (CGRP) receptor antagonists[e.g., αCGRP-(8-37)], interleukin receptor antagonists, (e.g.,Lys-D-Pro-Thr), phospholipase inhibitors including PLA₂ isoforminhibitors (e.g., manoalide) and PLCγ isoform inhibitors (e.g.,1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione),lipooxygenase inhibitors, (e.g., AA 861), prostanoid receptorantagonists including eicosanoid EP-1 and EP-4 receptor subtypeantagonists and thromboxane receptor subtype antagonists, (e.g., SC19220), leukotriene receptor antagonists including leukotriene B₄receptor subtype antagonists and leukotriene D₄ receptor subtypeantagonists, (e.g., SC 53228), opioid receptor agonists, includingμ-opioid, δ-opioid and κ-opioid receptor subtype agonists, (e.g., DAMGO,sufentanyl, fentanyl, morphine, PL 017, DPDPE, U50,488), purinoceptoragonists and antagonists including P_(2X) receptor antagonists andP_(2Y) receptor agonists, (e.g., suramin, PPADS), adenosine triphosphate(ATP)-sensitive potassium channel openers, (e.g., cromakalim,nicorandil, minoxidil, P 1075, KRN 2391, (−)pinacidil), neuronalnicotinic agonists (e.g., (R)-5-(2-azetidinylmethoxy)-2-chloropyridine(ABT-594), (S)-5-(2-azetidinyl-methoxy)-2-chloro-pyridine (S-enantiomerof ABT-594), 2-methyl-3-(2-(S)-pyrrolidinyl-methoxy)-pyridine (ABT-089),(R)-5-(2-Azetidinylmethoxy)-2-chloropyridine (ABT-594),(2,4)-Dimethoxy-benzylidene anabaseine (GTS-21), SBI-1765F, RJR-2403),3-((1-methyl-2(S)-pyrrolidinyl)methoxy)pyridine (A-84543),3-(2(S)-azetidinylmethoxy)pyridine (A-85380), (+)-anatoxin-A and(−)anatoxin-A (1R)-1-(9-Azabicyclo[4.2.2]non-2-en-2-yl)-ethanoatefumarate, (R,S)-3-pyridyl-1-methyl-2-(3-pyridyl)-azetidine (MPA),cystisine, lobeline, RJR-2403, SIB-1765F, GTS-21, ABT-418),α₂-adrenergic receptor agonists [e.g., clonidine, dexmedetomidine,oxymetazonline,(R)-(−)-3′-(2-amino-1-hydroxyethyl)-4′-fluoro-methanesulfoanilide(NS-49), 2-[(5-methylbenz-1-ox-4-azin-6-yl)imino]imidazo line(AGN-193080), AGN 191103; AGN 192172,5-bromo-N-(4,5-dihydro-1H-imidazol-2-yl)-6-quinoxalinamine (UK14304),5,6,7,8-tetrahydro-6-(2-propenyl)-4H-thiazolo[4,5-d]azepin-2-amine(BHT920), 6-ethyl-5,6,7,8-tetrahydro-4H-oxaazolo[4,5-d]azepin-2-amine(BHT933), 5,6-dihydroxy-1,2,3,4-tetrahydro-1-naphyl-imidazoline(A-54741)], mitogen-activated protein kinase (MAPK) inhibitors (e.g.,4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole,[4-(3-iodo-phenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-1H-imidazole],[4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole],[4-(4-fluoro-phenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole],2′-Amino-3′-methoxy-flavone), soluble receptors (e.g., tumor necrosisfactor (TNF) soluble receptors, interleukin-1 (IL-1) cytokine receptors,class I cytokine receptors, and receptor tyrosine kinases),corticosteroids (e.g., cortisol, cortisone, prednisone, prednisolone,flurdrocortisone, 6α-methylprednisolone, tramcinolone, betamethasone,dexamethasone) and local anesthetics (e.g., benzocaine, bupivacaine,chloroprocaine, cocaine, etiodocaine, lidocaine, mepivacaine, pramoxine,prilocaine, procaine, proparacaine, ropivacaine, tetracaine, dibucaine,QX-222, ZX-314, RAC-109, HS-37).

Suitable alternate or additional spasm inhibitory agents includeserotonin receptor antagonists (e.g., amitriptyline, imipramine,trazodone, desipramine, ketanserin, tropisetron, metoclopramide,cisapride, ondansetron, yohimbine, GR127935, methiothepin,oxymetazoline), tachykinin receptor antagonists including neurokinin₁receptor subtype antagonists (e.g., GR 82334, CP 96.345, RP 67580) andneurokinin receptor subtype antagonists (e.g., MEN 10.627, L 659.877,(±)—SR 48968), adenosine triphosphate (ATP)-sensitive potassium channelopeners, (e.g., cromakalim, nicorandil, minoxidil, P 1075, KRN 2391,(−)pinacidil), nitric-oxide donors, (e.g., nitroglycerin, sodiumnitroprusside, SIN-1, SNAP, FK 409 (NOR-3), FR 144420 (NOR-4),endothelin receptor antagonists, (e.g., BQ 123, FR 139317, BQ 610) andanticholinergics, including antimuscarinics (e.g., ditropan,tropicamide, cyclopentolate, scopolamine, atropine, homatropine andoxybutynin), antinicotinics (e.g., trimethaphan, macamylamine,pentolinium, pempidine and hexamethomium) and first generationantihistamines (e.g., diphenhydramine).

Methods of Use Periprocedural Delivery

Local perioperative delivery of the compositions of the presentinvention are expected to preemptively inhibit pain, inflammation andsmooth muscle spasm otherwise associated with urological procedures. Thecompositions of the present invention act on molecular targets, i.e.,receptors, enzymes and ion channels, that initiate pain, inflammationand spasm pathways and mechanisms. The present invention employs localperiprocedural delivery to inhibit these pathophysiologic processes atthe time they are initiated. For example, multiple proinflammatorypeptides stimulate the release of PGE₂ from bladder tissue within thefirst five minutes of exposure, as shown in the examples below. Solelypostprocedurally administered therapeutic agents can only take effectafter these processes have commenced.

Local Delivery

Local delivery of drugs in accordance with the present invention permitsthe utilization of a much lower dosage than would be needed if the samedrugs were administered systemically (e.g., orally, intravenously,intramuscularly, subcutaneously) to achieve the same predetermined locallevel of inhibitory effect in the urinary tract. The focused, localdelivery of the present invention results in a significantly lowerplasma level of the drug than would result from systemic delivery of thedrug to achieve the same predetermined local level of inhibitory effectin the urinary tract, thereby reducing the potential for undesirablesystemic side effects. Local delivery permits the inclusion in thecompositions of the present invention of drugs such as peptides that arenot susceptible to systemic delivery due to degradation during first-and second-pass metabolism.

Local delivery of drug compositions in accordance with the presentinvention provides for an immediate and certain therapeuticconcentration at the local urinary tract site, which is not dependent onvariations in metabolism or organ function. A constant concentration ofthe drugs can be maintained during the period of delivery of thecomposition during the procedure.

Urological Procedures

The compositions of the present invention can be locally deliveredbefore, during and/or after cystoscopy, i.e., the endoscopic examinationof the urethra and bladder through a cystoscope inserted into the lowerurinary tract for purposes of examining the urinary tract structures,preferably periprocedurally during such procedures. The compositions ofthe present invention may also be used before, during and/or after(preferably periprocedurally during) other diagnostic, interventional,medical and surgical procedures performed in conjunction withcystoscopy, by insertion of surgical instruments through the cystoscope,such as for the removal of tissue for biopsy, removal of growths,removal of foreign bodies, bladder or kidney stone removal, placement,removal and manipulation of urethral stents, transurethral resection ofbladder tumors (TURBT), treatment of tumors with electrocautery or laseror local chemotherapeutics, treatment of bleeding in the bladder or torelieve obstructions in the urethra.

The compositions of the present invention can be locally delivered tothe urinary tract before, during and/or after ureteroscopy, i.e., theendoscopic examination of the ureters and renal tissues through anureteroscope inserted through the urethra and bladder and into a ureterfor purposes of examining the urinary tract structures, preferablyperiperatively during such procedures. Ureteroscopy is often performedfor the drawing of urine samples from each kidney, the placement,removal and manipulation of ureteral stents, as part of the treatmentfor kidney stones, or to place a catheter in the ureter for a retrogradepyelography, and the compositions of the present invention can bedelivered before, during and/or after such procedures, preferableperiprocedurally during such procedures. A basket or other instrumentemployed via the ureteroscope can be used to capture the stone, thestone may be broken up by laser or shock wave lithotripsy through theureteroscope, or the ureteroscope may be employed to displace a lodgedstone back into the kidney for subsequent breaking up and passage, suchas by using a laser or extracorporeal shock wave lithotripsy (ESWL).

The compositions of the present invention are suitably locally deliveredto the urinary tract before, during and/or after procedures thattypically result in ureteral spasm, such as kidney stone removal usinglaser treatment, cystoscopy, ureteroscopy or lithotripsy, and preferablyperiprocedurally during such stone removal procedures.

The compositions of the present invention may also be locally deliveredto the urinary tract before, during and/or after (preferablyperiprocedurally) urological procedures that cause thermal trauma totissue in and/or associated with the urinary tract. These include lasertreatment to fragment stones or ablate tissue, microwave ablation oftissue (e.g., transurethral microwave thermotherapy (TUMT) to removeprostatic tissue), radiofrequency ablation of tissue (e.g.,transurethral needle ablation (TUNA) to remove prostatic tissue),electrocauterization or vaporization of tissue or cryoblation of tissue.

The compositions of the present invention may also be locally deliveredto the urinary tract before, during and/or after (preferablyperiprocedurally) urological procedures employing a laser for tissueresection, including Holmium: yttrium-aluminum-garnet (Ho:YAG),neodymium:yttrium-aluminum-garnet (Nd:YAG) andpotassium-titanyl-phosphate (KTP) “green light” laser therapies. Suchlaser procedures may include the treatment of benign prostatichyperplasia (BPH) and bladder tumors, by way of non-limiting example.

The ketoprofen composition, calcium channel antagonist and ketoprofencombination composition and the preferred ketoprofen and nifedipinecombination composition of the present invention may also be locallydelivered to the urinary tract before, during and/or after (preferablyperiprocedurally) transurethral resection of the prostate (TURP).

In addition to transurethral procedures such as those discussed above,the compositions of the present invention may also be suitably employedfor local delivery during other minimally invasive urologicalprocedures. These include, by way of example, the transrectal ortransperitoneal delivery of the compositions of the present invention tothe prostate and surrounding anatomic structures during implantation ofradioactive seeds and seed spacers to treat prostate cancer orprostatitis, and the transrectal or transperitoneal delivery of thecompositions of the present invention to the prostate to treatprostatitis.

The compostions of the present invention are suitably locally deliveredto the urinary tract before, during and/or after (preferablyperiprocedurally) procedures that standardly include irrigation, such asTURP, transurethral incision of the prostate (TUIP), laserprostatectomy, cystoscopy, ureteroscopy and other procedures in whichirrigation is used to aid visualization by removing blood and tissuedebris from the operative field. The compositions of the presentinvention can be added to the irrigation solution standardly used insuch procedures, e.g., saline, distilled water, lactated Ringer'ssolution, glycine, sorbitol, manitol, sorbital/manitol, at dilutelevels, with no change to the urologist's standard procedure beingrequired.

The compositions of the present invention can also be locally deliveredby coating ureteral stents, uretheral stents, catheters, radioactiveseeds, seed spacers or other implantable devices or surgicalinstruments, or impregnating or otherwise incorporating the therapeuticagents into the body of stents, catheters, radioactive seeds, seedspacers or other implantable devices or surgical instruments constructedfrom a polymeric material or mesh. Techniques for coating devices withdrugs and impregnating devices with drugs are well known to those ofordinary skill in the art, and coatings or polymeric materials may bedesigned to permit the drugs (e.g., a COX inhibitor and a calciumchannel antagonist) to begin releasing into the urinary tract uponimplantation and continuing for a period of time following implantation.

Formulation

One aspect of the invention is directed to a composition including acyclooxygenase inhibitor and a calcium channel antagonist, preferablyketoprofen and nifedipine, which are dissolved in an aqueous solutionfor parenteral delivery, preferably for intravesicular delivery.Alternately such compositions can be manufactured in a lyophilized formand then reconstituted with an aqueous solvent prior to administration.

The cyclooxygenase inhibitor and calcium channel antagonist are suitablyincluded in a molar ratio (cyclooxygenase inhibitor:calcium channelantagonist) of from 10:1 to 1:10, preferably from 5:1 to 1:5, morepreferably from 4:1 to 1:1, and most preferably 3:1. Similarly, in apreferred composition ketoprofen and nifedipine are suitably included ina molar ratio (ketoprofen:nifedipine) of from 10:1 to 1:10, preferablyfrom 5:1 to 1:5, more preferably from 4:1 to 1:1, and most preferablyapproximately (i.e., +/−20%) 3:1.

For compositions formulated to be delivered locally in a liquid carrier,the cyclooxygenase inhibitor such as ketoprofen is suitably included ata concentration (as diluted for local delivery) of no more than 500,000nanomolar, preferably no more than 300,000 nanomolar, more preferably nomore than 100,000 nanomolar and most preferably less than 50,000nanomolar. The calcium channel antagonist such as nifedipine is suitablyincluded at a concentration (as diluted for local delivery) of no morethan 200,000 nanomolar, preferably no more than 100,000 nanomolar, morepreferably no more than 50,000 nanomolar and most preferably less than25,000 nanomolar.

The compositions of the present invention may be formulated in anaqueous or organic solvent, but preferably are formulated in an aqueoussolvent. When using aqueous solutions, an additional solvent or solvents(i.e., cosolvents or solubilizing agents) may suitably be included toaid in dissolution of the drugs. Examples of suitable solvents includepolyethylene glycol (PEG) of various molecular weights (e.g., PEG 200,300, 400, 540, 600, 900, 1000, 1450, 1540, 2000, 3000, 3350, 4000, 4600,6000, 8000, 20,000, 35,000), propylene glycol, glycerin, ethyl alcohol,oils, ethyl oleate, benzyl benzoate, and dimethyl sulfoxide (DMSO). Apreferred cosolvent for the compositions of the present invention isPEG, most preferably PEG 400.

In a further aspect of the present invention, the composition includesketoprofen and nifedipine in an aqueous solution including at least onestabilizing agent. The term stabilizing agent is used herein to refer toan agent that inhibits degradation of the active pharmaceuticalingredients and/or extends the duration of stability of the solutionwhen stored under either refrigerated (e.g., 2-8° C.) or ambienttemperature conditions, and includes both anti-oxidants and chelatingagents. The solution may also suitably include one or more cosolvents orbuffering agents. Preferably the aqueous ketoprofen and nifedipinesolution includes one or more antioxidants as stabilizing agent(s), acosolvent and a buffering agent. The preferred ketoprofen and nifedipinesolution formulation is stable when stored at between 2° C. and 25° C.for a period of at least six months, preferably one year, morepreferably two years, most preferably longer than two years, and can bereadily diluted with standard urologic irrigation solutions for localintravesicular delivery during urological procedures.

Examples of suitable antioxidants for use as stabilizing agents in thecompositions of the present invention include water soluble antioxidantssuch as sodium bisulfite, sodium sulfite, sodium metabisulfite, sodiumthiosulfate, sodium formaldehyde sulphoxylate, ascorbic acid,acetylcysteine, cysteine, thioglycerol, thioglycollic acid, thiolacticacid, thiourea, dithithreitol, and glutathione, or oil solubleantioxidants such as propyl gallate, butylated hydroxyanisole, butylatedhydroxytoluene, ascorbyl palmitate, nordihydroguaiaretic acid andα-tocopherol. A preferred stabilizing agent for the present invention ispropyl gallate. When included in an aqueous composition, a cosolvent isincluded solubilizing oil soluble antioxidants such as propyl gallate. Apreferred aqueous ketoprofen and nifedipine composition of the presentinvention includes PEG 400 as a cosolvent and propyl gallate as astabilizing agent, and may more preferably also include a secondstabilizing agent such as a water soluble antioxidant, most preferablysodium metabisulfite. A suitable range of concentrations forantioxidant(s) is typically about 0.001% to about 5%, preferably about0.002% to about 1.0%, and more preferably about 0.01% to about 0.5%, byweight of the composition.

Because of the involvement of divalent cations in catalyzing oxidationreactions, the inclusion of a chelating agent as a stabilizing agent maybe useful in the compositions of the present invention. Examples ofsuitable chelating agents for use in the compositions of the presentinvention include the various salts of ethylenediamine tetraacetic acidsalts (EDTA), β-hydroxyethylenediaminetriacetic acid (HEDTA),diethylenetriamine-pentaacetic acid (DTPA) and nitrilotriacetate (NTA).

The compositions of the present invention suitably include a bufferingagent to maintain pH. Examples of suitable buffering agents forinclusion in the compositions of the present invention include aceticacid and its salts, citric acid and its salts, glutamic acid and itssalt and phosphoric acid and its salts. Citric acid also has the abilityto chelate divalent cations and can thus also prevent oxidation, therebyserving two functions as both a buffering agent and an antioxidantstabilizing agent. A preferred aqueous ketoprofen and nifedipinecomposition of the present invention includes citric acid (such as inthe form of sodium citrate) as a buffering agent and antioxidant, and ina more preferred composition also includes PEG 400 as a cosolvent andpropyl gallate and sodium metabisulfite as stabilizing agents.

The compositions of the present invention may also include additionalexcipients and adjuvants. Excipients may include a preservative toprotect against microbial growth, especially for multiple-dosecontainers. Suitable excipients include antimicrobial agents such asbenzyl alcohol, chlorobutanol, thimiserol, methyl paraben and propylparaben. Excipients may also include a surfactant to reduce surfacetension and thereby facilitate wetting for dissolution. Examples ofsuitable surfactants include polyoxyethylene sorbitan monooleate andsorbitan monooleate. Excipients may also include tonicity adjustmentagents to render the solution iso-osmotic with physiologic fluids.Examples of suitable tonicity agents include sodium chloride, sodiumsulfate, mannitol, glucose, sucrose, trehalose, and sorbitol. Additionalexcipients may include a colorant to impart color, such as FD& C No. 1blue dye, FD&C No. 4 red dye, red ferric oxide, yellow ferric oxide,titanium dioxide, carbon black, and indigo tar pigments.

TABLE 1 Exemplary Ketoprofen/Nifedipine Composition for Deliveryto theUrinary Tract (Stock Solution Concentrations Prior to Dilution)Exemplary Ingredient Function Concentration/Amount Ketoprofen COXinhibitor 7.63 mg/ml (30 mM) Nifedipine CA channel antag. 3.46 mg/ml (10mM) Sodium citrate Buffered solvent 20 mM solution aqueous solution (pH6.2 ± 0.5) PEG 400 Solubilizing agent 60% PEG 400:40% Sodium (cosolvent)citrate soln. (v:v) Sodium Antioxidant 0.02% metabisulfite (stabilizer)Propyl gallate Antioxidant 0.01% (stabilizer)

The above concentrated solution is diluted, such as at a ratio of1:1,000 (v:v) with standard irrigation solution such as saline orlactated Ringer's solution. The final dilute solution from the aboveexemplary formulation thus includes 0.06% PEG40, 0.00005% sodiummetabisulfite and 0.00001% propyl gallate (all by volume). The activeingredients are present in the final dilute solution at concentrationsof 0.00763 mg/ml (30,000 nM) for ketoprofen and 0.00346 mg/ml (10,000nM) for nifedipine.

EXAMPLES

The present invention may be illustrated by the following studiesdemonstrating the effects of ketoprofen and other cyclooxygenaseinhibitors, nifedipine and combinations of these agents in urologicalmodels, and demonstrating the stability of certain formulations of suchcompositions.

Example I The Effect of COX Inhibitors on Bradykinin Induced PGE₂Production in Rat Bladders

The following studies evidence that bradykinin induces immediateprostaglandin E₂ (PGE₂) production in the bladder, and demonstrate theeffects of cyclooxygenase inhibitors on this process. Bradykinin waschosen as the activating agonist for testing in this system because itsactions on the rat bladder tissue system have been well characterizedand because its role as a proinflammatory agent in acute pathophysiologyhas been studied. Bradykinin is also known to stimulate contraction ofsmooth muscle of the bladder when delivered intravesically by activationof B1 and B2 receptor subtypes.

1. Introduction

Acute, localized inflammatory responses in the lower urinary tract,including spasm, are triggered by surgical trauma. In response to tissueinjury, multiple inflammatory mediators, including bradykinin andSubstance P(SP) are released into the bladder. Exogenous application ofthese pro-inflammatory peptides or activation of bladder nerves cantrigger the production of prostaglandins (PGs) in the bladder. The aimof this study was to characterize the time course of production of PGsin response to an inflammatory mediator and evaluate the effects ofCOX-1/COX-2 inhibitors on bladder tissue contractility in vitro and invivo. The rat bladder tissue strip system represents a well establishedsystem for characterization of the pharmacological actions on numerousagents on smooth muscle bladder contractility [Edwards, G., et al.,Comparison of the Effects of Several Potassium-Channel Openers on RatBladder and Rat Portal Vein In Vitro, Br. J. Pharmacol. 102:679-80(1991); Birder, L., et al., β-adrenoceptor Agonists StimulateEndothelial Nitric Oxide Synthase in Rat Urinary Bladder UrothelialCells, J. Neurosci. 22:8063-70 (2002)].

2. Bladder Strip Contractility Method

Isolated bladder smooth muscle strips of 1×2×15 mm dimension wereobtained from Wistar derived male or female rats weighing 275±25 g thatwere sacrificed by CO₂ overexposure. Each strip was placed under 1 gtension in a 10 ml bath containing Krebs solution with 1 μM enalaprilicacid (MK-422), composition (g/l): NaCol 6.9, KCl 0.35, KH₂PO₄ 0.16,NaHCO₃ 2.1, CaCl₂ 0.28, MgSO 4.7, H₂O 0.29, (+)Glucose 1.8, pH 7.4bubbled with 95% O₂/5% CO₂ at 32° C. Each strip was connected to anisometric transducer (Harvard, #50-7293) and two-pen recorder andallowed to equilibrate for 60 minutes. Before starting the experiment,mounted tissues were validated for acceptance by challenge with 100 μMof methoxamine to obtain a minimum of 1 g tension, which was consideredas 100%. Qualified tissues were washed repeatedly every 15 minutes for60 minutes. A cumulative contraction-response curve to bradykinin wasthen generated through application of 3 concentrations of bradykinin(0.01 μM, 0.1 μM and 1 μM) at 1 minute intervals for a total of 3minutes. The tissue was subsequently washed periodically until tensionreturned to baseline value. Two hours later, the ability to inhibit thebradykinin cumulative dose response (0.01 μM, 0.1 μM and 1 μM) after a10 minute pretreatment with ketoprofen was determined. Eachconcentration of test substance was tested in four separatepreparations.

Results

FIG. 2 illustrates the cumulative concentration-response curves ofnormal animals to the agonists bradykinin and SP. The EC₅₀ forbradykinin was 8.5 nM and for SP was 6.5 nM. This provided a validatedsystem for testing the effects of the inhibitory activity of NSAIDs (COXinhibitors).

FIG. 3A illustrates bradykinin concentration-response curves produced inthe presence of 0.25, 1.0, 2.5 and 10 μM ketoprofen. The maximal agonistresponse could not be determined experimentally for all concentrationsof ketoprofen, although curve-fitting using a standard Hill equationrevealed no change in maximal response at saturating agonistconcentrations. Schild analysis was used to calculate the pA₂ value of7.26 for ketoprofen, equivalent to a K_(D) for ketoprofen at its site ofaction of 5.52×10⁻⁸ M (see FIG. 3B). This finding demonstrates that thepotency for inhibition in this tissue assay system is quite comparableto values obtained from direct enzyme inhibition assays.

3. PGE₂ Determination Methods

The release of PGE₂ from urinary bladder strips into 10 ml of tissuebath was measured using a specific enzyme immunoassay (EIA) according tothe manufacturer's instructions (Amersham Pharmacia Biotech) for thebasal, bradykinin-induced and COX inhibitor treatment plusbradykinin-induced samples. The COX inhibitors tested were ketoprofen,flurbiprofen, 5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonyl)thiophene(i.e., DUP-697) and1-[(4-methysulfonyl)phenyl]-3-tri-fluoromethyl-5-(4-fluorophenyl)pyrazole(i.e., SC-58125). One mL of fluid was collected from the 10 mL tissuebath after 10 minutes of bradykinin challenge for PGE₂ determination.Samples were frozen immediately and stored at −4° C. until assay. Thebladder strips were dried gently by blotting and were then weighed.Results are expressed as picograms of PGE₂ released per milligramtissue.

Results

FIG. 4A illustrates that bradykinin rapidly induces the formation ofPGE₂ in rat bladder tissue strips within the first minutes ofstimulation and reaches a maximum within 30 minutes. The t_(1/2) forformation was about 7.5 minutes. FIG. 4B illustrates the rapid kineticsof PGE₂ formation detected within the first ten minutes.

Ketoprofen inhibition of bradykinin-induced bladder strip contractionwas closely correlated with inhibition of PGE₂ formation, as shown inTable 2. Non-selective COX-1/COX-2 inhibitors were found to be effectivein blocking bradykinin-stimulated PGE₂, while COX-2 selective agentswere not effective. This corresponds to a lack of COX-2 inhibitoractivity under bradykinin-induced normal cystometry parameters.

TABLE 2 Inhibition of Bradykinin (BK)-induced Contraction with COXInhibitors BK-induced Contraction BK-induced PGE₂ Drug IC50 (μM) IC50(μM) Ketoprofen 0.97 0.58 Flurbiprofen 24.8 1.65 DUP-697 >25 >25SC-58125 >25 >25

FIG. 1 (described previously) provides a model for action ofprostaglandin activity. Activation of bradykinin receptors on urothelialcells may produce PGs in the urothelium, which in turn may activatebladder nerves (C-fiber and M fibers) to affect bladder contractilityand control micturition reflexes. Ketoprofen inhibits formation of PGE₂.

4. In Vivo Rat Cystometry Model Methods

The rats were anesthetized with urethane at 1.2 g/kg i.p. in 5 ml/kg. Apolyethylene catheter (PE50) was implanted into the bladder for salineor acetic acid infusion through a 3-way stopcock. A pressure transducerwas connected for measurements of intravesical pressure. Warm (37° C.)saline was infused into the bladder at a constant rate of 16.7 ml/min (1ml/hour) until cystometry became stable (no less than 60 minutes).Thereafter, 0.2% acetic acid was infused into the urinary bladder.Aspirin (10 mg/kg i.v.) and vehicle were administered intravenously viaa PE-10 catheter in the femoral vein at 5 minutes after infusion ofacetic acid was started and at the end of first micturition cycle.Dunnett's test was applied for comparison between the time before andafter test substance or vehicle treatment. To ascertain differencesbetween the test substance and the vehicle control group, an unpairedStudent's t test was used. Differences are considered significant atp<0.5.

Results

FIG. 5A illustrates that intravenous aspirin (10 mg/kg) produced agradual time-dependent inhibition of the acetic acid induced reductionin the intercontraction interval (ICI), and FIG. 5B illustrates theparallel changes in bladder capacity. Threshold pressure and micturitionpressure were not affected by aspirin treatment (data not shown).

6. Discussion

These studies demonstrate that PGE₂ is rapidly produced in rat bladdertissue following stimulation with bradykinin and that its formation isinhibited by a 10 minute pre-incubation with ketoprofen. Non-selectiveCOX-1/COX-2 inhibitors were demonstrated simultaneously to have blockedthe rapid production of PGs in bladder tissue and tissue contractility.Aspirin and other non-selective COX-1/COX-2 inhibitors effectivelyinhibited cystometric changes induced by intravesical acetic acidstimulation. These studies suggest that delivery of ketoprofen to theurinary tract may be therapeutically beneficial for periproceduralbladder hyperactivity.

Example II Effects of Ketoprofen and Nifedipine Individually onBradykinin Induced Contractility in Rat Bladder Tissue Strips

The purpose of this study was to characterize the effects of ketoprofen,a non-selective COX-1/COX-2 inhibitor, and nifedipine, an L-type Ca²⁺channel antagonist, on agonist-stimulated rat bladder contractilityusing bradykinin as a stimulating agonist.

1. Methods

Ketoprofen USP and nifedipine USP were dissolved in DMSO prior todilution to the final concentration. Bladder tissue strips from Wistarderived rats were prepared, transduced and equilibrated using thebladder strip contractility method described in Example I above. Assayedtissue was incubated with the test drugs for 10 minutes beforeactivities were determined.

A cumulative contraction-response curve to bradykinin was generatedthrough application of 7 bradykinin concentrations in 3-fold incrementsranging from 0.001 μM to 1 μM at 1 minute intervals for a total of 7minutes to establish the maximal 100% control response. The tissue wassubsequently washed periodically until tension returned to baselinevalue. In 24 separate tissues, similar bradykininconcentration-responses were carried out in the presence of eachrespective test compound (ketoprofen: 0.25 μM, 1 μM, 2.5 μM and 10 μM;nifedipine: 0.125 μM, 0.5 μM, 1.25 μM and 5 μM) following a 10 minincubation period. Tissue strips were always used in pairs for the studyof the action of the antagonist (bradykinin) alone and in the presenceof a concentration of antagonist (ketoprofen or nifedipine). Schildplots were obtained using computer software (Pharmacology CumulativeSystem, Version 4) and pA₂ values were determined.

2. Results

Nifedipine was found to exhibit a noncompetitive type of antagonism uponbradykinin-induced contractile responses in the in vitro rat bladderpreparation. This was shown by a depression of the maximum agonistresponse and a small non-parallel rightward shift of the agonistconcentration response curves (FIG. 6). In contrast, as previouslydescribed in Example I, increasing concentrations of ketoprofen (0.25-10μM) produced a series of concentration-response curves (see FIG. 3A) inwhich the EC₅₀ agonist response moved progressively to higherconcentrations of bradykinin (shift to the right of over 2 orders ofmagnitude) with no apparent effect on maximal tension. This pattern ofinhibition is consistent with a competitive mechanism for ketoprofen andwas further analyzed by Schild regression analysis.

For nifedipine, the criteria for application of the Schild regressionanalysis were not met due to the noncompetitive pattern of inhibition.Even the lowest concentration of nifedipine (0.125 μM) resulted in alarge reduction in the agonist response (to about 50% of maximum). Thesestudies of ketoprofen and nifedipine reveal two very different patternsof inhibition of bradykinin-stimulated contractile tension.

Example III Effects of Ketoprofen and Nifedipine Combination onBradykinin Induced Contractility in Rat Bladder Tissue Strips

The present study evaluated the effects of nifedipine and ketoprofenadministered in combination on the contractile tension response in a ratbladder tissue strip model.

1. Methods

Bladder tissue strips from Wistar derived rats were prepared, transducedand equilibrated using the bladder strip contractility method describedin Example I above with transduced strips being allowed to equilibratefor 45 minutes. In order to avoid effects of bradykinin receptordesensitization from the cumulative dosing protocol, two tissue stripswere collected from each animal. The control group consisted of 12strips and 54 strips were used for the treatment groups.

Before starting the experiment, each pair of tissue strips was qualifiedby treating with 0.03 μM bradykinin to determine if the initialdifference in maximal contraction between strips was within +/−15%.Following this procedure, qualified tissues were washed repeatedly every15 minutes for 60 minutes. Cumulative concentration-response curves weregenerated by application of bradykinin to establish maximal response.For the control group (n=12), a cumulative concentration-response curveto bradykinin was then generated through application of nineconcentrations from 0.1 nM to 1.0 μM in 3-fold steps at one minuteintervals, for a total of nine minutes to establish the maximal 100%control response. Response curves for the treatment groups involvedpre-incubation of the bladder tissue for a period of ten minutes (n=6),followed by generation of bradykinin cumulative dose-response curves byapplication of 12 concentrations of bradykinin (0.1 nM-30 μM).

The concentration range that was chosen for each of the active agentswas based upon results from prior in vitro pharmacological studies ofeach single agent described in Examples I and II above. Those studiesshowed that ketoprofen in the 0.3-3 μM range had measurable effects onthe EC₅₀ for bradykinin activation. Ketoprofen at 3 μM was near maximalin its ability to shift the EC₅₀ of the bradykinin activated responsecurves on muscle contractility. Similarly, prior testing of nifedipineidentified a range of concentrations (0.05-5 μM) effective at inhibitingbradykinin induced tension. A factorial design characterized the effectsof nine different two-drug combinations of ketoprofen and nifedipine atthe following concentrations of (i) ketoprofen: 0.3, 1.0, or 3.0 μM; and(ii) nifedipine: 0.1, 0.3 or 1.0 μM. The treatment groups (groups 2-10)tested are summarized in Table 3 below:

TABLE 3 Ketoprofen-Nifedipine Combinations Tested Group Ketoprofen Conc.(μM) Nifedipine Conc. (μM) 1 (Control) — — 2 0.3 0.1 3 0.3 0.3 4 0.3 1.05 1.0 0.1 6 1.0 0.3 7 1.0 1.0 8 3.0 0.1 9 3.0 0.3 10  3.0 1.0

The bradykinin concentration-response data was fit to a variable slopesigmoidal equation, also known as the 3-parameter logistic response(3PL) function, to obtain the maximal tension, EC₅₀, and Hill slope inwhich the bottom of the curve was fixed at 0. The force of contractionin the presence of inhibitors was expressed as a percentage of themaximum bradykinin effects observed within the same strip beforeaddition of an inhibitor.

2. Results

The experimental data for all curves allowed curve fitting to accuratelydefine the maximal tension and EC₅₀ values. The control curve in FIG. 7showed that BK concentration-dependently increased the force ofcontraction with a pEC₅₀ of 8.14 or 72 nM (n=12 strips). A moderate Hillslope of 0.65 characterized the activation curve. All furthercontraction data was expressed as a percentage of the maximum bradykinineffect obtained from a set of 12 tissue strips without any tension andwithout any antagonist present.

The results for nine distinct combinations of nifedipine and ketoprofenused to inhibit bradykinin-induced bladder contraction are shown in thefollowing three tables and three figures. At the lowest concentration ofnifedipine and ketoprofen tested, 0.1 μM and 0.3 μM respectively, 38%reduction of the maximal control tension was observed (Table 4).Increasing concentrations of ketoprofen (1.0 and 3.0 μM) in the presenceof the same concentration of nifedipine further decreased the maximalcontractile tension such that only 30 and 23.4% of the control tensionremained, respectively. All concentration-response curves for bradykininshifted to the right in the presence of nifedipine and ketoprofen(0.3-3.0 μM), with the greatest effect seen at the highest ketoprofenconcentration. This combination was accompanied by a 1.0 log unit shiftin the pEC₅₀ versus control. The changes in the EC₅₀ parameter did notappear correlated with changes in maximal tension. The results arepresented graphically in FIG. 7, which compares the control group andthe group having a constant concentration of 0.1 μM nifedipine with arange of concentrations of ketoprofen. The percent of contraction foreach drug combination is expressed as the percent of the maximalresponse for the bradykinin control. The overall pattern of inhibitionpredominantly reflects a substantial decrease in maximal tension,demonstrating that the combination of nifedipine and ketoprofen acttogether in combination mechanistically in a non-competitive antagonistmanner towards bradykinin-induced contractions.

TABLE 4 Concentration-Response Curve Fitted Parameters for 0.1 μMNifedipine (NIF) plus 0.3-3.0 μM Ketoprofen (KET) T_(max) Log EC₅₀ HillSlope Concentration of Drug Est. SEM Est. SEM Est. SEM Control 100.004.17 −8.14 0.09 0.65 0.07 0.1 μM NIF + 0.3 μM KET 62.11 3.90 −7.61 0.180.69 0.16 0.1 μM NIF + 1.0 μM KET 30.13 1.92 −7.95 0.17 1.02 0.35 0.1 μMNIF + 3.0 μM KET 23.38 2.24 −7.02 0.24 0.62 0.16 Est. = Estimated SEM =Standard error of the mean T_(max) = Maximal tension determined by curvefitting

In the presence of 0.3 μM nifedipine, increasing concentrations ofketoprofen present in the combination treatment resulted in aprogressive decrease in the maximal tension, from 36.4 to 16.0%.Combinations utilizing the higher concentration of nifedipine (0.3 μM)resulted in a greater reduction in the maximal tension relative to thecorresponding concentrations of ketoprofen in the presence of 0.1 μMnifedipine. The maximal tension levels for 0.3 μM nifedipinecombinations were determined for three combinations, in concentrationratios of nifedipine:ketoprofen of 1:1, 1:3.3 and 1:10. The curve fittedparameters data obtained are presented in Table 5.

Comparison to data corresponding to ketoprofen concentrations in Table 4shows that in all cases, greater reductions in the maximal tension wereassociated with the greater nifedipine concentration. The greatestchange was evident at the lowest ketoprofen concentration, 0.3 whichdecreased from 62.11 to 36.41%. The higher concentrations of ketoprofenresulted in an even greater reduction in tension, such that only 16%remained at 3.0 μM. Associated with these changes in tension, a similarshift in the EC₅₀ relative to the control of 0.5 log units was evidentfor all ketoprofen concentrations at this nifedipine concentration, ascan be seen in FIG. 8. As in the case of 0.1 μM nifedipine, no apparentdifferences between the EC₅₀ values for this concentration of nifedipinewere evident. Small differences in the Hill slopes for bradykininagonist responses over the range of inhibitor concentrations were notsignificant. The effect of increasing ketoprofen concentrations in thecombination treatment on the concentration-response curves is similar tothe graph of the data at 0.1 μM nifedipine and various ketoprofenconcentrations. These graphical data also show the non-competitivenature of the antagonism of the BK-response, which is seen for thecombination at this higher concentration of nifedipine.

TABLE 5 Concentration-Response Curve Fitted Parameters for 0.3 μMNifedipine (NIF) plus 0.3-3.0 μM Ketoprofen (KET) T_(max) Log EC₅₀ HillSlope Concentration of Drug Est. SEM Est. SEM Est. SEM Control 100.004.17 −8.14 0.09 0.65 0.07 0.3 μM NIF + 0.3 μM KET 36.41 3.84 −7.49 0.290.66 0.24 0.3 μM NIF + 1.0 μM KET 28.88 2.24 −7.69 0.22 0.72 0.21 0.3 μMNIF + 3.0 μM KET 15.96 1.82 −7.59 0.29 0.94 0.51 Est. = Estimated SEM =Standard error of the mean T_(max) = Maximal tension determined by curvefitting

The overall shapes of the response curves observed in the presence of1.0 μM nifedipine were similar at all concentrations of ketoprofen. At1.0 μM nifedipine, the maximal tension levels were less than thecorresponding values for 0.3 μM nifedipine (Table 6 and FIG. 9), and themagnitude of the additional change due to the presence of ketoprofen isless relative to lower concentrations of nifedipine. The EC₅₀ valueswere uniformly shifted about 0.51 units for all ketoprofenconcentrations and were not correlated with maximal tension. Thispattern was consistent with observations at all other combinationconcentrations. A small additional increase in the inhibition of maximaltension due to the change from 1.0 to 3.0 μM ketoprofen was observed atthis highest concentration of nifedipine. At the highest concentrations(1.0 μM nifedipine plus 3.0 μM ketoprofen), 89% inhibition of thecontrol tension level was achieved.

TABLE 6 Concentration-Response Curve Fitted Parameters for 1.0 μMNifedipine (NIF) plus 0.3-3.0 μM Ketoprofen (KET) T_(max) Log EC₅₀ HillSlope Concentration of Drug Est. SEM Est. SEM Est. SEM Control 100.004.20 −8.14 0.09 0.65 0.07 1.0 μM NIF + 0.3 μM KET 21.60 1.00 −7.50 0.120.93 0.20 1.0 μM NIF + 1.0 μM KET 12.30 1.00 −7.48 0.20 0.96 0.35 1.0 μMNIF + 3.0 μM KET 10.80 0.80 −7.34 0.16 1.08 0.37 Est. = Estimated SEM =Standard error of the mean T_(max) = Maximal tension determined by curvefitting

3. Response Surface Analysis

The concentrations of the two agents (nifedipine and ketoprofen) used inthis combination experiment represent independent variables. The maximaltension is an effect that results from the combination and is theresponse variable of primary interest for the response surface analysis.The relationship between the drug combinations and the response variablecan be represented in a three-dimensional plot in which theconcentrations are plotted as Cartesian coordinates in the x-y-plane,and the response variable (e.g., maximal tension) is plotted as thevertical distance above the planar point. The collection of spatialpoints plotted in this way provides a view that represents the combinedconcentration-response relationship. The advantages of this experimentaldesign method include the fact that the biological response measured isnot limited to a specific response (effect) level of the system. In thisway, a number of fixed-ratio concentration combinations can be testedover a wide range of concentrations to define the interaction efficacyof the two drugs.

As in the case of single drug concentration-biological effectrelationships in which a smooth curve (or line) may be best fit to thedata according to a specific model, a smooth surface may be fit to thedata in a three-dimensional plot of a two-drug combinationconcentration-response relationship. This surface represents theadditivity or interaction of the combination. The graph of this responsesurface becomes the reference surface for viewing actual combinationeffects and allows the visualization and prediction of effects inregions of the curve for which no data could be generated.

A standard response surface analysis was performed on the estimatedmaximal tension and EC₅₀ values. The response surface model was fittedusing as a response variable the tension values at the highest agonistconcentration on each individual dose response curve, which was thetension corresponding to 30 μM BK. FIG. 10 shows the fitted responsesurface for the reduced model as a function of ketoprofen and nifedipineconcentration. The combination response curve drops steeply withincreasing concentrations of both ketoprofen and nifedipine. The surfacebecomes fairly flat as the maximal response is obtained asconcentrations approach 1 μM nifedipine+3 μM ketoprofen. Theconcentration combination that results in 90% maximal inhibition of theeffect of bradykinin is 3 μM of ketoprofen+1 μM of nifedipine.

4. Discussion

The study of this Example III evaluated the effects of nifedipine incombination with ketoprofen using bradykinin as an agonist to stimulatesmooth muscle contraction. Bradykinin was used in the rat bladder tissuestrip assay system (Examples I-III) to serve as an endogenous mediatorof contraction. The overall pattern of inhibition seen with allcombinations of nifedipine and ketoprofen concentrations wascharacteristic of non-competitive antagonism. Nifedipine, which preventsthe influx of calcium ions through the cell membrane by acting on L-typevoltage-dependent channels, attenuates the bradykinin receptor activatedcontraction of smooth muscle without directly inhibiting the receptor.As a single agent, nifedipine inhibition was shown above (Example II) tocause a reduction in the maximum bradykinin responses that were notaccompanied by statistically significant changes in the agonist potencyof the remaining response.

This study revealed the surprising finding that the magnitude of theinhibition is greatly enhanced by the addition of ketoprofen at thelowest nifedipine concentration tested and is evident at allconcentrations of the combinations tested. At low concentrations ofnifedipine, this inhibition is more than additive, i.e., synergistic innature. In contrast, ketoprofen treatment alone at the sameconcentrations was observed to not decrease maximal contractile tension,with no significant effect on the EC50 values for the nine combinationsand no concentration dependence upon ketoprofen. Thus, this synergisticinteraction on maximal tension and lack of strong effect upon the EC50was an unexpected result based on the study of ketoprofen action whentested as a single agent in this test system.

Taken together, these data indicate that the effects of theproinflammatory agonist, bradykinin, can be in part mediated by thesimultaneous activation of L-type calcium channels and the induction ofarachidonic metabolites that together augment smooth muscle contraction.While not wishing to be limited by theory, this effect may be due to apositive feedback loop that operates at a cellular and tissue level.Prostaglandins generated intracellularly as a result of bradykininreceptor activation may move to the extracellular environment, wherethey may interact and in turn activate prostanoid receptors subtypes.There are at least four known prostanoid receptor subtypes, termed EP1,EP2, EP3 and EP4. Of these subtypes, EP1 receptors are believed to becoupled through G proteins to stimulation of phophoinositide hydrolysisand/or PLC-independent influx of calcium. EP1 receptors have beenpreviously identified in smooth muscle, where they can function tomediate contractile activity. Hence, the discovery of the combinedsynergistic actions of ketoprofen and nifedipine on contractile activitymay be a result of simultaneous blockade of calcium mobilization and theconcurrent inhibition of a positive-feedback loop involving PGE₂ drivenactivation of prostanoid receptors.

In conclusion, each combination of nifedipine and ketoprofen showed agreater inhibition of maximal bradykinin-induced contraction compared toeither drug alone in the rat bladder tissue strip assay. Furthermore,the multiple combinations of nifedipine and ketoprofen tested allowed aresponse surface analysis to define optimal concentrations. A fixedratio combination containing 3.0 μM ketoprofen and 1.0 μM nifedipine wasidentified that produced ˜90% inhibition.

Example IV Inhibition by Nifedipine and Ketoprofen of MultipleAgonist-Induced Contractile Tension and Release of PGE₂ in Rat BladderTissue

The objective of this study was to evaluate the effects of ketoprofenand nifedipine on rat bladder contractility and agonist-stimulated PGE₂production using multiple agonists. Bradykinin, substance P, histamineand ATP are endogenous mediators that can be released as part of theacute inflammatory response and activate bradykinin receptors (B1 and B2subtypes), tachykinin receptors (NK₁₋₃) and histamine receptors (allsubtypes) and purinergic P2X and P2Y receptors, respectively.Carbamylcholine is an agonist that may activate muscle and neuronalnicotinic acetylcholine subtypes or muscarinic acetylcholine receptorssubtypes (M₁₋₅) present in the bladder, while methoxamine is specificfor α₁-adrenergic receptors. The first objective was to evaluate theeffect of ketoprofen (10 μM) and nifedipine (1 μM) individually, each ata fixed concentration, on contractile tension induced by each of the sixagonists (bradykinin, substance P, carbamylcholine, methoxamine,histamine and ATP) in the rat bladder tissue strip model. The secondobjective was to determine the amount of PGE₂ released from the bladdertissue in response to stimulation by each agonist in the presence ofeither ketoprofen or nifedipine during the same test conditions employedto measure contractile smooth muscle tension.

1. Methods

Bladder tissue strips from Wistar derived rats were prepared, transducedand equilibrated using the bladder strip contractility method describedin Example I above. Either 10 μM ketoprofen or 1.0 μM nifedipine waspre-incubated individually with the tissue for a period of 10 minutesprior to stimulation with the following agonists at a concentrationequivalent to its respective ED₇₅ for stimulation of tension: 0.03 μMbradykinin; 0.03 μM substance P; 3.0 μM carbochol; 30 μM methoxamine; 25μM histamine; and 20 μM ATP. Antagonist activity for a givenconcentration of an antagonist (nifedipine or ketoprofen) was determinedas the ability of that concentration of the antagonist to reduce thenoted agonist-induced (e.g., 0.03 μM bradykinin-induced) response by 50percent or more (50%). Each concentration of antagonist was tested infour separate tissue preparations.

The effects of the two drugs on PGE₂ release in response to multipleagonists was compared using the same 10 min pre-incubation protocol anda subsequent 30 min incubation period with agonist in the presence ofthe test compound. PGE₂ produced after 30 minutes of treatment with eachagonist (e.g., 0.03 μM bradykinin) in the absence and presence of thetest compounds was determined. An initial 1.0 ml sample was taken fromthe tissue bath after a 30 minute incubation with the agonist.Subsequently, the tissue was washed using 10 ml of Krebs solution every15 minutes for a 2 hour period. The test compound was added andpre-incubated for a period of 10 minutes prior to re-challenge with thesame agonist. After an additional 30 minute period in the presence ofthe test antagonist and agonist, 1.0 ml was removed from the bath foranalysis. The release of PGE₂ from urinary bladder strips was measuredusing a specific enzyme immunoassay (EIA). Samples were frozenimmediately and stored at −4° C. until assay. The bladder strips weredried gently by blotting, and then weighed. Results are expressed aspicograms of PGE₂ released per milligram tissue.

2. Results

All of the agonists investigated stimulated contraction of the bladdertissue strips, independent of their mechanism of action, demonstratingthat multiple mediators can increase bladder smooth muscle contractiletension. Nifedipine (1 μM) produced a significant inhibition (>67%) ofeach agonist-induced increase in contractile tension (FIG. 11). Thecontractile response to bradykinin was affected by both nifedipine andketoprofen (81% inhibition and 67% inhibition, respectively). Incontrast, the increase in contractile tension induced by substance P,carbamylcholine and ATP was not affected by ketoprofen. Ketoprofen alsoonly slightly reduced the tension for methoxamine and histamine (<25%).

Bradykinin evoked the largest increase in PGE₂ relative to the otheragonists tested. This evoked release was effectively inhibited byketoprofen (81%) but minimally affected by pre-treatment with nifedipine(12%) (FIG. 12). Thus, the extent of inhibition of smooth muscle tensionby nifedipine was not linked to agonist-induced PGE₂ responses and wasdistinct from the effect of ketoprofen. The absolute bladder levels ofPGE₂ produced in response to stimulation by other GPCR agonists wereabout 10-fold less than those seen with bradykinin.

In summary, the current study indicated that an increase in smoothmuscle contractile tension can be induced by a variety of GPCR agonistsin bladder tissue. Moreover, a common signaling mechanism for theseagents is mediated in part through activation of L-type Ca²⁺ channels inthe rat urinary bladder. Nifedipine's inhibition of L-type Ca²⁺ channelssuggests an effective mechanism for inhibition of numerouspathophysiological mediators that can lead to increased smooth musclebladder tension associated with spasm or hyperactivity. Ketoprofeninhibited bradykinin-stimulated PGE₂ production and release from thebladder while nifedipine did not exhibit an effect on this response.Thus, nifedipine and ketoprofen act through distinct mechanisms toinhibit smooth muscle contractile tension and release ofpro-inflammatory prostaglandins in bladder tissue.

Example V Effect of Ketoprofen and Nifedipine on Rat Bladder Function inan Acetic-Acid Overactive Bladder Model

The primary objective of this study was to measure the effect ofketoprofen and nifedipine during intravesical, local delivery to femalerats with overactive bladder function caused by perfusion with salinecontaining 0.2% acetic acid (acidified saline). Perfusion of 0.2% aceticacid through the bladder is known to rapidly induce an acuteinflammatory state that is reflected in functional changes in bladdercystometry.

1. Methods

The method used in the current study represents an adaptation of awidely used acetic acid-triggered rat model of hyperactive bladder. Inthis model, acute inflammation of the bladder is produced by using 0.2%acetic acid in saline as the bladder perfusion fluid and cystometryunder anesthesia is performed after a recovery period from the surgicalprocedure. A regular interval of voiding cycles can be seen for severalhours after the initial stabilization period occurs. A bladder catheterconnected to an infusion pump was used to deliver the drug solutionsdirectly to the bladder at a constant, defined rate.

The animals were anesthesized and bladder catheters were surgicallyimplanted to allow irrigation of the test agents. The followingcystometry parameters were monitored: intercontraction interval (ICI),trigger pressure (TP), micturition pressure (MP) and micturition volume(MV) using a Med Associates Cystometry Station and software program.Only rats that displayed normal and stable cystometry profiles duringthe preliminary saline-infusion stage (not less than 15 min of baselinestabilization followed by 7 regular representative ICI intervals) wereincluded in the study. Following the saline period, the rat bladder wasinfused with test agent in saline containing 0.2% acetic acid for 20 minfollowed by the collection of 7 representative ICI intervals foranalysis. Due to the fixed concentrations of the irrigation solutionsemployed in the study and the use of constant perfusion rates for fixedconstant times, a fixed, uniform dose of each agent was delivered to allanimals.

Groups of female rats were administered ketoprofen at selectedconcentrations (0.01-25 μM) alone or nifedipine at selectedconcentrations (0.1-10 μM) alone. Five to seven animals were normallytested in each group. Acidified saline served as the control. Allinfusion solutions were prepared fresh on the day of the experimentbefore use. For each of the test agents, three distinct bladderirrigation periods were employed: 1) baseline (saline only) for 1 hour;2) drug in saline only for 15 minutes; and 3) drug in 0.2% acidifiedsaline for 1 hour.

2. Results

In the control animals, baseline levels of bladder contractions inresponse to a constant irrigation rate of 0.1 μl/min saline wereestablished during the first hour following surgery. The time betweencontractions (ICI, seconds) and peak micturition pressure (MP, mm Hg)appeared to vary somewhat between animals but was fairly constant withinanimals following stabilization. Following the addition of the 0.2%acetic acid to the perfusion buffer, rapid contractions appeared,resulting in a significant decrease in the ICI. Increases in contractilepressure accompanied the shortening of time between the bladdercontractions in many cases as well. These changes in functional bladderresponses could be routinely measured following perfusion of the bladderwith acidified saline, as shown in FIG. 13. A 40-50% shortening of theICI was typically seen in the control group in response to the 0.2%acetic acid irrigation (mean % ICI=58.4%±6.8%, n=8).

Inclusion of ketoprofen in the irrigation buffer leads to aconcentration-dependent inhibition of the shortening of the ICI (FIG.14). Complete inhibition was seen at approximately 3 μM ketoprofen andhigher concentrations tended to go above 100% (data not shown).

Inclusion of nifedipine in the irrigation buffer also leads to aconcentration-dependent inhibition of the shortening of the ICI (FIG.15). Complete inhibition was not seen but maximal effects appeared to beat 1 μM nifedipine, and higher concentrations tended to plateau atapproximately 75% of baseline.

Example VI Pharmacokinetics of Absorption of a Nifedipine and KetoprofenCombination in a Rat Bladder Saline Model

The primary objective of this study was to measure systemic plasmalevels of ketoprofen and nifedipine during and after the intravesical,local delivery of a combination of these drugs to rats. A secondaryobjective of this study was to determine the rate of appearance ofketoprofen and nifedipine when administered individually or incombination. Finally, a third objective of this study was to evaluatethe effects of local drug delivery on the rat bladder tissue content ofthe pro-inflammatory mediator, PGE₂, following surgical trauma to thebladder and subsequent intravesical perfusion of each agent or thecombination.

1. Methods

The study included three main treatment groups of animals: a combinationof both ketoprofen (10 μM) and nifedipine (10 μM); ketoprofen (10 μM)alone; and nifedipine (10 μM) alone. A bladder catheter connected to aninfusion pump was used to deliver the drug solutions directly to thebladder at a constant, defined rate.

For each of the three drug treatment groups, three distinct bladderirrigation periods were employed that were defined by the bladderperfusion solution for each period: 1) baseline (saline only) for 1hour; 2) drug in saline only for 1 hour; and 3) post-drug saline periodfor 30 minutes (min). The animals were anesthetized and the dome of thebladders were surgically implanted with a catheter to allow perfusion ofthe test agents with an infusion pump at a constant flow rate of 100μl/min. During period 1, saline was the perfusion fluid used and noplasma samples were collected. Starting at period 2, plasma samples werecollected at time points of 0, 15, 30, 45 and 60 min following perfusionof test agents. Subsequent to 60 min of perfusion with test agents, onlysaline was perfused for an additional 30 min and two additional timepoints at t=75 and 90 min were collected to determine the acutepost-perfusion phase of test agents. Due to the fixed concentrations ofthe irrigation solutions employed in the study and the use of constantperfusion rates for fixed constant times, a fixed, uniform dose of eachagent was delivered to all animals.

Whole blood samples were collected into K₂ EDTA tubes at the specifiedcollection times. The volume of whole blood collected was approximately0.2 mL per sample. The blood was spun in a centrifuge and the plasmatransferred into polypropylene tubes. Plasma samples were stored frozenat −80° C. until shipment for analysis. Rats were euthanized by CO₂inhalation and the bladder was rapidly dissected and frozen in liquidnitrogen and stored frozen at −80° C. until assayed for tissue PGE₂content.

The combination of ketoprofen and nifedipine was formulated inaccordance with an aspect of the invention to include ketoprofen (10mM), and nifedipine (10 mM) in a 60% polyethylene glycol 400 (PEG400):40% water solvent base, including 50 mM sodium citrate buffer for apH 7.5 solution in a 5 mL glass vial. Immediately prior to use, thecombination solution was diluted in the standard irrigation fluid at aratio of 1:1000 such that the final concentrations of the active drugsdelivered directly to the bladder were each 10 μM. For theseexperiments, a fixed concentration ratio of 1:1 nifedipine:ketoprofenwas chosen, and final concentrations of 10 μM for each agent weremaintained in the irrigation buffer.

2. Results

The study demonstrated a very low level of systemic absorption ofketoprofen following perfusion of the bladder with saline containing 10μM ketoprofen for 60 min. In four out of six rats, a narrow range ofC_(max) between 4.3-5.8 ng/ml was seen at 60 min. At the 60 min timepoint, the perfusion with 10 μM ketoprofen was stopped and normal salineirrigation was continued for an additional 30 min period. For the groupof four out of six rats which showed peak plasma levels of about 5ng/ml, plasma levels decreased at 75 and 90 minutes following cessationof ketoprofen perfusion. Delayed absorption during the 75-90 minuteinterval was observed in the other two animals in the ketoprofen-onlygroup.

For comparison, the ketoprofen levels were also determined for thecombination of ketoprofen and nifedipine. The increase in systemicplasma levels was approximately linear over time during the initial 60minute drug perfusion phase and the absolute mean plasma levels of 9.3ng/ml (n=6) at 60 minutes were well below the acceptable therapeuticdaily dose of ketoprofen. As in the case of the ketoprofen only group,the perfusion with the combination was stopped and normal salineirrigation was continued for an additional 30 minute period. The meanketoprofen values for all animals (n=6) were not significantly differentat 60, 75 and 90 minutes.

A comparison of the mean plasma ketoprofen results are presentedgraphically in FIG. 16 for the ketoprofen-only group and the combinationgroup. The mean values (and standard error of the means, SEMs) clearlyshow the constant plasma levels for the combination after 60 minutes.Although small differences are apparent in the earliest phase of thetime-course, no significant differences were observed either in the peaklevels or in the absorption kinetics for the ketoprofen plasma levels inthe combination group versus the ketoprofen alone group after 30minutes, or in the peak levels, indicating that no apparentketoprofen-nifedipine drug interactions were present.

The overall kinetic profile observed for nifedipine was similar to thatobserved for ketoprofen. In the nifedipine-only plasma group, nifedipineplasma levels increased linearly in 5/6 animals and some delayedabsorption was observed in only 1/6 animals. The C_(max) plasma level inthe nifedipine group was in the range of 10.6-16.0 ng/ml at 60 minutesfor 5/6 animals. The mean peak plasma levels observed at 60 minutes werebelow the acceptable mean peak levels of 79±44 ng/ml that are obtainedin man as a result of an oral therapeutic daily dose of nifedipine.

The increase in nifedipine systemic plasma levels from the combinationof ketoprofen and nifedipine also exhibited a linear increase withincreasing time for the initial 60 minute drug perfusion period. TheC_(max) plasma levels in the combination group had a mean value of 18.2ng/ml and values ranged from 8.2-34.6 ng/ml at 60 minutes for all sixanimals. The mean peak plasma levels observed at 60 minutes are aboutone fourth the mean peak levels that are obtained as a result of oraltherapeutic daily dose of nifedipine.

As shown in FIG. 17, a comparison of the mean peak plasma concentrationsof nifedipine (and plotted SEMs) shows the similar linear increase thatoccurs during the initial perfusion phase of intravesical delivery. Nosignificant differences in nifedipine plasma levels were seen in thenifedipine only group when compared with the nifedipine and ketoprofencombination drug product group.

At the end of the 90 min bladder perfusion period, bladders wereharvested from the animals and subsequently the entire bladder wasanalyzed for PGE₂ content using an enzyme immunoassay system. Data shownin FIG. 18 are expressed as the mean of PGE₂ using units of pg/mgprotein±the standard error of the mean from six animals per treatmentgroup. When animals were treated with nifedipine, bladder tissue PGE₂levels of 421±97 pg/mg protein (n=6) were observed compared tostatistically significantly (p<0.05) lower levels in the presence ofonly ketoprofen or ketoprofen and nifedipine in the combinationtreatment group, 83±22 (n=6) and 115±63 pg/mg (n=5), respectively. Nostatistically significant differences were seen between the ketoprofentreatment or the combination treatment groups. In summary, ketoprofentreatment alone or treatment with the combination during bladderperfusion significantly inhibited PGE₂ formed in the whole bladderrelative to the nifedipine treatment group.

3. Discussion

Using a method of intravesical perfusion for local drug delivery, thedrugs tested in this study were directly in contact with the absorptivesite within the bladder. The continuous perfusion maintained constantdrug concentrations of either ketoprofen, nifedipine or the combinationwithin the bladder during the period of drug delivery. Under theseconditions, minimal systemic exposure to the drugs occurred in femalerats during a 1 hour intravesical perfusion. Low levels of each drugwere detectable within the first 15 min interval measured, andabsorption progressed gradually as an approximately linear function overtime of drug perfusion for each agent.

The locally delivered drugs and drug combination were exposed to thestructures of the bladder, including the uroepithelium, C-fiberafferents, efferents and smooth muscle. The data obtained in the studyshow that this action is local and cannot be ascribed to systemic effectthat could be mediated through central nervous system mechanisms becausethe initial levels in the plasma for both drugs tested are so low.

Comparison of the plasma levels for each agent tested to known humanlevels associated with normal oral dosing reveals the magnitude of thedifference observed. In the combination treatment group, the maximallevels for ketoprofen were about 400-fold less than the peak plasmalevels (C_(max)) in humans that are associated with the acceptabletherapeutic daily dose of ketoprofen (rat mean ketoprofen plasma levelof 9.29±2.13 ng/ml at 60 min). For comparison, the accepted daily meanpeak C_(max) for a single 200 mg ketoprofen tablet (a single oral dose)is 3900 ng/ml. Similarly, peak levels observed for nifedipine wereapproximately 15 ng/ml to 25 ng/ml. The maximal levels (C_(max))typically occurred at the end of 60 min drug perfusion period or withinthe following 30 min sampling period. For comparison with known plasmalevels from conventional oral dosing, the accepted daily C_(max) for asingle 10 mg immediate release nifedipine tablet is reported to be 79±44ng/ml. Systemic exposure was comparable for ketoprofen plasma levelswhether administered alone or with nifedipine. Similarly, nifedipineplasma levels were comparable whether administered alone or withketoprofen.

This study also determined the PGE₂ content of the bladder for each ofthe treatment conditions in the study. An additional finding ofsignificance is the long-lasting effect of ketoprofen that was measuredin the assay of whole bladder PGE₂ levels. The low concentrations of theketoprofen treatment alone or the combination treatment during bladderperfusion significantly inhibited PGE₂ formed in bladder tissue relativeto the nifedipine treatment group. The PGE₂ bladder tissue levels in thepresence of ketoprofen were not significantly different from thosefollowing treatment with the combination. Because delivery of the drugwas stopped at 60 minutes in this study, and then saline was used toirrigate for an additional 30 minutes, this showed that PGE₂ inhibitionremained active in the post-drug delivery period. Thus, ketoprofendemonstrated an extended period of anti-inflammatory activity in thismodel of local, intravesical drug delivery.

Example VII

The purpose of this study was to evaluate the solubility of ketoprofenand nifedipine in aqueous liquid solution formulations.

1. Methods

Three ketoprofen and nifedipine combo liquid formulations, identified asF3/1, F10/3, and F30/10, were prepared according to the compositionshown in Table 8 below. In all three test formulations, 50 mM sodiumcitrate aqueous buffer was used. The target solubility ofketoprofen/nifidipine for F3/1, F10/3, and F30/10 were 3 mM/1 mM, 10mM/3 mM, and 30 mM/10 mM respectively.

TABLE 8 Solubility Results for Three Nifedipine and KetoprofenCombination Formulations PEG400% Approximate (v/v) Target SaturationFormu- Added Solubility Solubility Formula- lation to (Ketoprofen/(Ketoprofen/ tion ID Buffer Buffer Nifedipine) Nifedipine) F3/1  50 mMNa 35% 3 mM/1 mM 4.5 mM/1.5 mM citrate buffer pH 5.5 F10/3 50 mM Na 50%10 mM/3 mM 15 mM/4.5 mM citrate buffer pH 5.5  F30/10 50 mM Na 60% 30mM/10 mM 45 mM/15 mM citrate buffer pH 5.5

2. Results

In order to achieve complete dissolution of both actives, ketoprofen andnifedipine, in the formulatins, different percentages of PEG 400, 35%v/v PEG 400 (F3/1), 50% v/v PEG 400 (F10/3), 60% v/v PEG 400 (F30/10),were used as a cosolvent. With the assistance of PEG 400 as asolubilizing agent, the approximate saturation solubility of ketoprofenand nifedipine in all three formulations was approximately 1.5× of theirrespective target solubility. The solubility results in Table 8 clearlyindicate that PEG 400 is a suitable solubility enhancing agent for bothdrugs when it is desired to prepare highly concentrated combinationsolution formulations.

Example VIII

The purpose of this study was to evaluate the stability of exemplarycombination ketoprofen and nifedipine aqueous liquid solutionformulations.

1. Methods

Four exemplary ketoprofen and nifedipine combination solutionformulations, identified as F1 to F4, were prepared according to thecomposition shown in Table 9. In all four formulations, theconcentrations of the active drugs were 3 mM for Ketoprofen and 1 mM forNifedipine. All four formulations employed sodium citrate buffer (pH5.5) with a 35% v/v of PEG 400. The ionic strength of the buffer usedwas 50 mM for F1 and F2, and 20 mM for F3 and F4. No antioxidant wasadded to the virgin formulation F1, while 0.05% propyl gallate, 0.02%sodium metabisulfite, and 0.05% propyl gallate plus 0.02% sodiummetabisulfite were added to the combination formulations F2, F3, and F4respectively.

TABLE 9 Tested Nifedipine and Ketoprofen Combination FormulationsFormula- Drug Concentration Formulation Antioxidants tion ID(Ketoprofen/Nifedipine) Vehicle Added F1 3 mM/1 mM 50 mM NaCitrate None(pH 5.5) w/35% v/v PEG 400 F2 3 mM/1 mM 50 mM NaCitrate 0.05% Propyl (pH5.5) w/35% gallate v/v PEG 400 F3 3 mM/1 mM 20 mM NaCitrate 0.02% Sodium(pH 5.5) w/35% metabisulfite v/v PEG 400 F4 3 mM/1 mM 20 mM NaCitrate0.05% Propyl (pH 5.5) w/35% gallate & v/v PEG 400 0.02% Sodiummetabisulfite

An isocratic high performance liquid chromatograph (HPLC) method wasused to quantify ketoprofen and nifedipine and their related substancesin these test solution formulations after storage for different periodsof time. Formulation samples were taken and diluted into mobile phase toobtain a final concentration of approximately 0.76 mg/mL to 2.54 mg/mLfor ketoprofen and approximately 0.35 mg/mL to about 1.15 mg/mL fornifedipine. Chromatographic conditions for the related substances assaywere as follows: (1) detection wave length: UV 241 nm; (2) column:Zorbax SB-C18, 5 μM, 4.6×150 mm; (3) column temperature: 30±1° C.; (4)flow rate: 1.0 mL/min; (5) injection volume: 20 μL; (6) run time: 27minutes.

2. Results

FIG. 19 shows an example chromatogram of the combination solutionformulation F1 after stressing by storing at 60° C. for 1 month. The twoactive ingredients, ketoprofen and nifedipine, have a retention time of24.19 minutes and 19.31 minutes respectively. There are four mainrelated substances with relative retention times (RRT) of 0.34, 0.58,0.75, and 0.87 relative to the nifedipine peak. This stability data issummarized in Table 10.

TABLE 10 Total related Substance (%) of Ketoprofen and Nifedipine inTested Formulations After Storage at Different Temperatures FormulationID Days 4° C. 25° C. 40° C. 60° C. F1 0 — 1.08 — — 14 1.17 4.17 8.83 281.49 3.39 6.69 16.45  F2 0 — 1.07 — — 14 1.57 — 2.89 3.63 28 1.89 5.463.18 4.75 F3 0 — 1.34 — — 14 1.76 2.86 4.54 8.39 28 2.11 3.51 5.4412.68  F4 0 — 0.28 — — 14 0.29 0.31 0.34 0.81 28 0.30 0.33 0.51 1.49

The stability data in Table 10 indicates that the chemical stability ofketoprofen and nefidipine, especially nifedipine, is significantlyimproved in the presence of a small amount of either propyl gallate(0.05% w/v) or sodium metabisulfite (0.02% w/v) at elevated temperaturessuch as 40° C. and 60° C., with this effect being unexpectedlypronounced for propyl gallate. When a small quantity of both propylgallate (0.05% w/v) and sodium metabisulfite (0.02% w/v) is added to F4,the stability of the two drugs at all temperatures is significantlyimproved when compared with the other three combination formulationswithout antioxidant or with one of the two antioxidants alone,suggesting additive or synergistic degradation inhibition effect of thetwo antioxidants.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes to the disclosedsolutions and methods can be made therein without departing from thespirit and scope of the invention. It is therefore intended that thescope of letters patent granted hereon be limited only by thedefinitions of the appended claims.

The embodiments of the invention in which an exclusive property orprivilege is claimed are defined as follows:
 1. A stable composition forus in inhibiting pain/inflammation and spasm, comprising a combinationof ketoprofen, nifedipine, propyl gallate and sodium metabisulfite in aliquid carrier, wherein the ketoprofen and nifedipine are included at aketoprofen to nifedipine molar ratio of between 10:1 to 1:10.
 2. Thecomposition of claim 1, wherein the liquid carrier comprises an aqueoussolvent.
 3. The composition of claim 2, wherein the composition furthercomprises polyethylene glycol 400 as a cosolvent.
 4. The composition ofclaim 1, wherein the composition further comprises a citric acid buffer.5. The composition of claim 1, wherein the ketoprofen and nifedipine areincluded at a ketoprofen to nifedipine molar ratio of between 5:1 to1:5.
 6. The composition of claim 5, wherein the ketoprofen andnifedipine are included at a ketoprofen to nifedipine molar ratio ofbetween 4:1 to 1:1.
 7. The composition of claim 6, wherein theketoprofen and nifedipine are included at a ketoprofen to nifedipinemolar ratio of 3:1 plus or minus 20%.